To construct a oligonucleotide microarray for the detection of five important pathogens including CMV、RV、HSVI、TB、EBV in central nervous system..METHODS : 70 mer oligo probes targeted the specific protein of 5 common pathogens of infectious diseases of the nervous system were designed for the fabrication of microarray. DNA was extracted with conmercilized kit. The target genes were labeled with biotin-dUTP by random primer extension method using the DNA as template. The labeled products were hybridized with the oligonucleotide microarray. After hubridization the microarray were binding with avidin-CY3. After scan with Scanarray 4000 confocal scanner, the fluorescent intensity was analyzed with Quantarray3.0 software. This oligonucelotide microarray system were used to detect clinical sample and compared with PCR method. RESULT: The specific probes for the pathogens are very specific for the detecting the presence of the pathogens. Clinical detection results showed the specificity and sensitivity of this microarray system are 95% and 83% respectively. There is no significant difference between our method and the routine PCR method.. CONCLUSION:The microaaray system for the detection of the major pathogen of central nervous system has the advantage of specific,sensitive and parallel detection. We can add pathogen probes according to the practical needs. It can not only be used for the diagnosis of central nervous system, but also canbe used for the detection of blood, urine, and other body fluid infection.
Objective To construct transcription regulatory network of liver-selective transcription factors(LSTF). Methods Tissue-selective Affymetrix probes set (3919 probes all together) were classified based on their functional categories. LSTF genes were then selected for further analysis. The 500-bp upstream sequences of all LSTF genes were extracted for predicting binding sites of known transcription factors (TFs). Transcription regulatory network was constructed using LSTF and related TFs as input. Results Four LSTF genes were obtained from functional classification and tissue selectivity screen, which share 4 upstream TFs in common as predicted by two programs. Transcription regulatory network among LSTF, predicted TFs and some other related genes demonstrates a fairly complicated topology, with ID2 and PROX1 centering in the network. Conclusions Based on existing literatures, we argue that ID2 and PROX1 genes may play important role in regulating LSTF genes transcription, because they are involved in hepatocyte growth and differentiation, etc.
We used differential adherence selection to enrich spermatogonial stem cells (SSCs)from pup mouse testis, and transfer them to recipients pre-treated with busulfan through rete testis. Two months after transplantation, recipient mouse testes were recovered and observed that the transplanted donor cells had colonized into recipient seminiferous tubules. At 113 day after transplantation when recipients were mated with wild-type females of same genetic background, offspring with the donor cells genetic information were produced. This study indicated that the testis cells selected by differential adherence selection not only contained enriched SSCs, but also maintained their stem cell activity，which allows them to colonize recipient testis and restore normal spermatogenesis.
Objective: To construct the eukaryotic expression vector for DINH and sC3d3 fusion gene and express it in vitro．Methods: The fragment of C3dwas amplified by RT-PCR and inserted into pMDT-18 plasmid and then sub cloned into eukaryotic expression vector pcDNA-DPPIS-DINH-mC3d3.The recombinant plasmid pcDNA-DPPIS-DINH-sC3d3 was transfected into BHK-21 cells and the expressed product was detected by Western blot. Results: Western blot demonstrated that DINH and sC3d3 existed in the cultural supernatant．Conclusion: The constructed DNA vaccine can be expressed in vitro, which may pave a way for further studies in animals.
Abstract ：A 120bp DNA fragment encoding antibacterial peptide of sarcophaga peregrina A was designed based on the amino acid sequence of antibacterial peptide and the biased codon usage of pastoris．Eight oligonucleotides were chemically synthesized，linked and then recombinant with α-factor gene , recombinanted gene was cloned into yeast expression vector pPIC9K．After restriction enzyme analysis and DNA sequencing，the recombinanted gene of antibacterial peptide was transfected the Pichia pastoris GSll5 strain．The positive clones screened by the phenotype were induced by methanol , the expression product was tested by SDS-PAGE and inhibit zone using E.coli SD and E.coli C3000 as tested strain. The results showed that antibacterial peptide gene was expressed in Pichia pastoris successfully． The expression product was secreted outside with the lead of α-factor signal and had strong antibacterial activity．
ABSTRACT AIM:Cloning two genes of heparin-binding domain of fibronectin into vector of insect expression and expressing two polypeptide in insect cell Sf9.Purifing polypeptide with Ni-NTA and Testing biological activity of polypeptide binding with heparin and antygen of FN with Western-blot.METHODS:The gene of Two heparin-binding domain of fibronectin were amplified from FNcDNA by high fidelity PCR. The aim DNA sequences were cloned into Bac-to-Bac HT Vector(AorB) to get recombinant plasmid. The recombinant plasmid was transformed into E.coli DH5α And the positive plasmid was selected throuth ampicillin resistance to get positive recombinant donor.After being sequenceed and determined right read frame,positive donor was transformed into MAX Efficiency DH10BacTM Competent E.coli to get positive bacmid (white colony )by gentamicin,kanamycin and tetracycline resistance with IPTG and X-gal.recombinant bacmid transformed into Sf9 cell and in it recombinant baclovirus was formed.Isolating baclovirus from supernatant of culture Sf9 cell and performing a viral plaque assay,then recombinant baclovirus were transformed into Sf9 cell again , polypeptide was expressed in it. Polypeptide in supernatant of insect cell lysate were purified with Ni-NTA colummn .Binding heparin and FN antygen activity of polypeptides were detected by western blot assay. Tt was determined in mice that rhFNHC-36 polypeptide antagonizes endotoxemia with thrombocytopenia. RESULTS: Two heparin-binding polypeptide of fibronectin were expressed in insect cell sf9.Expressed polypeptides can bind with heparin in vivo and have losted antygen of FN . The rhFNHC-36 polypeptide has function of antagonizing endotoxemia with thrombocytopenia in mice. CONCLUSION: Two heparin-binding polypeptide of fibronectin can be expressed in insect expression system.Expressed polypeptide can bind with heparin in vivo ,not with antygen of FN and have function of antagonizing endotoxemia with thrombocytopenia in mice. Exprssion ratio is low,more method must be found to elevate it.
The baculovirus has been widely used as a pesticide in many countries. However, baculovious has some shortcomings such as the speed and spectrum killing insects was slower and narrower than some chemical pesticides. Therefore, the scientists are making efforts to reconstruct baculovirus in order to get new type biological pesticides. Ecdysteroid UDP-glucosyltransferase, egt gene of baculovirus was responsible for the insect exuviate and an important gene. The paper mainly described how to extract SeMNPV genome DNA.The DNA could be successfully used to amplify the purpose gene by polymerase chain reaction(PCR) using SDS-proteinase K-saturated phenol methods，and then the egt gene was cloned from SeMNPV. The result showed that the positive high pure plasmids could be extracted with Alkali method and digested directly with endonuclease. It was known that ORF of SeMNPV egt was 1572bp long and encodes 523 amino acids through DNAstar software analysis. TATA boxes lies at 48~53 and 68~72bp of the 5'-nonencoding region upstream of a translational start site. Comparing with seven kinds of nuclear polyhedrosis virus and one kind of granulosis virus, it indicated that the SeMNPV egt gene is the highest match the Agrotis setegum nuclear polyhedrosis virus(AsNPV).It has 75% nucleotides and 79% amino acid identity. And some of amino acids which closely related to structure and function are very high conservative. Through egt protein phylogenetic tree analysis, it is known that baculovirus would be divided into two classes, one is NPV; and another is granulovirus. The work can lay the foundations for baculovirus genectic engineering in future.
Cre/loxP system is a recombinase system which origins from bacteriophage P1.It has become an important tool for genetically modified bioengineering because it is time-specific, tissue- specific，site-specific and efficient recombination.In this paper, the recombinant characteristics of the Cre/loxP system, the compositions and structure of cre recombinase，recombination mechanism and application in yeast, is reviewed.
To achieve high level expression of human transferring (hTF) and investigate the possible mechanism of the hTF expression, rabbit transferrin promoter (RP promoter) was cloned, and a hTF recombinant expression vector (δpcDNA3.1(-)-RP-hTF), was constructed in which hTF DNA was driven by RP promoter. The transient transfection experiments achieved a high hTF expression in BRL-3A and HC-11 cell lines, and two stably integrated cell clones were also screened out. Various concentrations of 5-aza-dC were added to the cell culture medium for the investigation of the effect on hTF expression. Real-time RT-PCR analysis showed that the RP promoter directed an efficient hTF expression, and 5-aza-dC increased hTF expression in a proportional manner. However, the extent of the increase in hTF expression differed between individual clones, suggesting that TF expression may relate to gene methylation.
objective: To prepare recombinant human polyamine oxidase (PAO) and polyclonal antibody of rabbit-anti-human PAO by gene recombination techniques. Methods: Human PAO cDNA was amplified by RT-PCR from total RNA of A549 cells. PAO cDNA was then cloned into pET-15b to construct PAO prokaryotic expression vector. The vector was transformed into E. coli. BL21(DE3) and induced to express PAO recombinant protein by IPTG. PAO was purified by Ni-NTA resin under denature condition and dialyzed to recover its native structure. After that, the enzyme activity was analyzed with chemical fluorescent method. PAO polyclonal antibody was prepared by using recombinant human PAO protein which was purified by polyacrylamide gel electrophoresis as antigen to intradermally inoculate and immunize rabbit. The antibody titer and specificity were determined by ELISA, Western Blot and Immune Cell Chemistry. Results: Purified and dialyzed recombinant human PAO has the enzymatic activity of quickly oxidizing N1-acetylspermine. The antibody prepared by recombinant PAO has high titer and specificity against human PAO. Conclusion: The methods for prokaryotic expression, purification of human PAO and preparing specific anti-PAO antibody were successfully established. These methods laid a foundation for the succedent functional research of PAO as an anti-tumor target.
PAP-C23, a kind of deleted mutant of pokeweed antiviral protein (PAP) gene, was amplified from mature pokeweed antiviral protein gene by PCR, then the amplified fragment was cloned into the expression vector PET101, and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3) strain. Induced with IPTG, the recombinant gene was expressed. SDS-PAGE analysis showed that the recombinant protein existed in the form of inclusion.The recombinant protein amount may accounted for 29.6% of the total inclusion proteins by optimizing the expression conditions. After recycled by cutting the gel which has been stained by KCL solution, the recombinant protein was grinded fully with a pestle in ice-bath and dissolved in phosphate buffered saline(PBS), and then added into same volume Freund's adjuvant incomplete. After emulsified completely, the mixed solution was used to inject rabbits, to prepare polyclonal antibody. Indirect enzyme linked immunosorbent assay(ELISA) showed that the titer of the antiserum was 1:1000. Western blot analysis showed that the antiserum could specifically react with expressed recombinant protein and nature PAP isolated from Phytolacca amercana, which also indicated that the recombinant PAP gene was expressed correctly in Escherichia coli.
Abstract Although BT insecticide had been widly applies in agricultural production , some problems still existed,like low yield and easier to lose activity.Polh protein that stable in natural circumstance is a protector for the virion. And more, the recombinant polyhedrin showed almost the same characteristics as the native baculoviral polyhedrin: it was rapidly solubilized under alkaline conditions and digested by alkaline proteases in the insect midgut. So our target is to investigate the use of the baculoviral Polh protein as a stable and protective fusion partner for the high expression of BT. Recombined BT cry3Aa with AcNPV Polyhedrin, construct the expression vector and made the recombined protein expressed in E.coli. Obtained the recombined protein engineering strain of cry3Aa and polyhedrin, and so laid foundation for the applied experimentation.
Present paper is a review on the metabolism, application and preparation of astaxanthin. Having astaxanthin synthesized and regulated genes expressed in fishes was suggested using state-of-the-art metabolic and genic engineerings that will benefit for the development and growth of the fish. The result can be applied in other hydro－animal.
Abstract: Objective Study the stimulative effects of different concentrations insulin and D-glucose on Toxoplasma gondii replication in 3T3-F442A cells and determination of its activity so as to establish the optimal growth condition of T. gondii. Methods The tachyzoite concentration was determined by enumeration in a haemocytometer (LEVY counting chamber). The survival of tachyzoites was examined using an MTT assay. Results were expressed as means ±S.E.M. Significance of the data was evaluated by Student's unpaired t-test. Results Insulin concentrations between 10－2 and 10－1 mg/ml combination of 4.5 g/l D-glucose in DMEM medium gave maximum stimulus to T. gondii replication. However, insulin, in the absence of D-glucose, had the least effect on T. gondii growth. D-glucose concentrations significantly affected the tachyzoite replication and appear to be indispensable for maintaining the host 3T3-F442A cells. Conclusion Insulin and D-glucose have a dose-responsive mitogenic effect on intracellular T. gondii replication and development in 3T3-F442A cells and lower concentrations of insulin have a significant positive effect on T. gondii growth though higher concentrations of insulin have a negative effect on T. gondii replication. However, insulin, in the absence of D-glucose, had the least effect on T. gondii growth.
The I phase flagellin antigen of the three strong pathogenic strains Salmonella paratyphi A, Salmonella typhimurium, Salmonella Choleraesuis strain SGSC2461 is H-1a, H-1i and H-1c respectively. Three pairs of primers were designed in order to amplify the specifical gene fragments of epitopes with the length of 1088bp, 633bp and 1056bp by using PCR amplification technique. Utilized Linker of the primer and PCR amplification technique to connect the three antigenic determinant gene sequence in series,so the recombinant gene of flagellum is 2747bp altogether. Constructed the prokaryotic expression vectors pET-28a-F of flagellin recombination gene, and transfered the plasmid into E.coli BL21 then induced protein expression by IPTG. SDS-PAGE electrophoretic analysis that interest protein weighted 95Ku was expressed. It optimized the expression conditions. The percent of interest protein, which had been induced in terminal density 1mmol/L of IPTG under 37℃, is about 11.2% through thin-layer scanning analysis.Cracked the bacteria with supersonic wave, and purified cytorrhyctes which contained interest protein. Immuned the foot pads of gynaec Guinea Pig eight weeks aged. The titer of antibody detected by indirect ELISA was 1:512000, and the sensitivity was 8μg/mL.This is a foundation of research of detection of the fusion protein in the late stage.
Aloe has been deeply researched recently because of its attracting function on medical treatment, hairdressing, health protection and edibility. It was investigated that DREB transcription factor were able to improve crop the tolerance to abiotic and biotic stresses. In this experiment, aloe stems were used as explants for OsDREB gene transformation mediated by Agrobacterium. PCR analyses demonstrated that OsDREB gene was successfully transferred into aloe genome with a transformation efficiency of 0.5%. Relationship between relative electrical conductivity and cold resistance was studied through positive and negative plants treated with the same low-temperature. The results of relative electrical conductivity in positive plants were higher than the negative plants. And the leaves of negative plants appear severe evidence of freeze injury with brown, withered and translucent, as the positive plants appear good growing condition.
Based on the previously reported sequence of soybean 1-aminocyclopr- opane-1-carboxylate synthase gene (GenBank Accession No.dq273840), three gene-specific reverse primers, CTA798 sp1, CTA798 sp-2, CTA798 sp3 were designed, and a DNA fragment about 600bp was obtained by TAIL-PCR. The transcription start site was localized at 62bp upstream of the start ATG identified by 5′-RACE. The obtained sequence was analyzed by PLACE and PLANTCARE program and showed putative ACC synthase gene promoter features: it contains the basic core elements of TATA-box and CAAT-box, as well as stress-induced elements: light-responsive element Box1, Box4 and elicitor-induced element W-box. Based on the binary vector pBI121, a plant gene expression vector carrying gusA gene under the putative ACC synthase gene promoter was constructed and introduced into Agrobacterium tumefaciens EH105. The transient expression of gusA gene was then observed by histochemical staining in tobacco leaves through the leaf disc transformation mediated by Agrobacterium tumefaciens, which revealed the obtained sequence has promoter activity.
To develop a fast and easy reproducible propagation system for Cyclamen persicum Mill. and study variation of somatic embryos, the tubers of seedlings were used as explants, and embryogenic callus were induced after cultures and subcultures. The embryogenic callus were used as inocula for establishment of suspension culture system. The regeneration of somatic embryos in high frequencies was observed after plating the cultures from solid and liquid medium on hormone-free medium.
The endo-β-1,3-glucanase from Schizophyllum commune Fr. was purified in three steps which comprised ammonium sulfate precipitation, DEAE-Sephadex A- 50 chromatography, and Sephadex G-100chromatography. The purified enzyme showed purification 14.60 fold, and an activity recovery of 11.8%. The optimal temperature and pH of the enzyme were 45 ℃ and 5 . 0 respectively . The β-1,3-glucanase was more stable at pH 4.5-5.5, and was relatively stable below 60 ℃. Zn2+、K+、Cu2+、Ag+、Hg2+、Ca2+had inhibitory effect on the enzyme activity; Zn2+, Ca2+, Fe2+ and Ba2+ could stimulate the activity
Carrageenase were produced by Bacillus amyloliquefaciens, which fermented with carrageen as inductor. The optimal condition of zymosis and degradation determined by viscosity change was achieved: liquid seed inoculate 2%, carrageen terminal concentration was 0.1% in the solid leavening, and degrading temperature of 50℃, pH 5, the enzyme quantity was 0.5：1（w/w）. Its molecular weight came down from 1296kDa to 1660Da.
After testing the resistance 0f streptomycin to GX-29，the spores treated with UV light and microwave were regenerated on agar plants containing 18.2μg/mL streptomycin. A number of streptomycin resistant (str) mutants were obtained. Then we acquired a high antibiotic-producing strain L8 through re-screening by batch fermentation.The activity of the mutant L8 increased by 1.94 times over the original strain．The high productivity could be inherited after several subcultures.
Aspergillus oryzae has received increasing attention as a source for homologous and heterologous protein production due to its ability to secrete large amounts of protein. The amount of enzymes secreted by Aspergillus oryzae in the solid-state culture greatly exceeds that secreted in the submerged culture..However, the mechanisms involved in protein production remain unknown. Studies have focused on the unfolded protein response in the endoplasmic reticulum (ER) because this step in protein maturation represents the most probable bottleneck in the protein secretory pathway .As of the present time, the UPR pathway has been identified in A. nidulans, T. reesei and A. niger , but UPR regulation under solid-state culture is still unknown.This study compared the expressional level of the hacAi between solid-state and submerged cultures through real-time PCR, to understand the differences in the UPR between the two culture conditions. The results show that the expressional level of the hacAi under solid-state cultivation of A. oryzae is higher than under submerged cultivation. And increased with low water activity.
We analyze the component of ion-exchange wastewater which can be produced in the process of manufacturing monosodium glutamate by this experiment, and attempt to use ion-exchange wastewater as the fermentation matrix to produce pullulan. The optimum conditions of culture are as follows: initial pH is 6.5,pullulan cells are transferred to 500ml conical flasks containing 150ml culture medium; inoculation is incubated according to 10% dosage; The culture medium is carried out in a rotary shaker incubator at 200rpm and at 28℃ for 7 days. As a result, 23.5g /L Crude pullulan can be obtained, and the conversion rate is 47%. By shucking off pigment through activated carbon column and then removing protein, the recovery rate of pullulan reaches 65%.
Nowadays, the synthesis of polyamidoamine denerimers includes "solid phase synthesis" and "liquid phase synthesis". A new thought of synthesis polyamidoamine denerimer by fall together this two advantage is introduced. Having glycin as the core, a new fan-shape polyamidoamine denerimers are synthesized. The Fluorescence and Release Property are being studied too. The dendrimer were then reacted with 1- acetic-5-fluorouracil to form dendrimer-5FU conjugates. Hydrolysis of the conjugates in a phosphate buffer solution (pH 7.4) at 37℃ will release free 5FU. The dendrimer seems to be a promising carrier for the controlled release of anti-tumor drugs.
Large-scale cultivation is an essential step towards the feasible production of baculovirus in insect cell cultures. Airlift reactors appear to offer considerable advantages over other insect cell culture systems. In order to evaluate the impact of reactor design on the behavior of insect cell cultures, the Helicoverpa Amrigera cell HzAm1 was cultivated in airlift reactors with different geometrical parameters. The ratio of downcomer to riser cross sectional area, the bottom clearance and the ratio of height to diameter of the reactor are proved to be important of the cell growth behavior. The influence of gas flow rate on the growth of HzAm1 cells cultivated in a selected reactor configuration was determined to find an optimal superficial gas velocity that renders sufficient oxygenation without any negative effects on the cellular viability. And the influence of the reactor design and superficial gas velocity on fluid circulation in the reactor was tested. Modifying the reactor design and the superficial gas velocity ,the maximum viable cell density could be elevated from 4.5×105to 7.7×105cells/ml，and also the cell livability is improved.
Gene trap vector pU17 can be integrated into chromosome DNA of human hepatoma cells randomly. After selected with G418, X-gal staining and PCR checking, several high X-gal expression cell colonies were obtained. A special Hepatitis B virus (HBV) polymerase full-length cDNA vector containing mutant loxP sites exchanged -geo DNA fragment of gene trap vector in these colonies with the Cre enzyme. Under rigorously selection of puromycin, a new cell line expressing HBp-His fusion protein was established. HBp protein could be identified well with the His-tag antibody. This hepatoma cell line might be a useful tool for preparation HBp protein antigen and analysis of its function.
The purification process to rapidly produce a large of plasmid had been developed and an in vivo electroporation device was made by our own. We evaluated delivery of plasmid vector that encode reporter Enhanced Green Fluorescent Protein (EGFP) into rabbit skeletal muscle. Reporter gene result showed that plasmid DNA transfer in muscle fibers was very efficient by the in vivo electroporation device. According to the result of rabbit intramuscular plasmid injection followed by the electroporation device, the female lambs and pigs were injected with myogenic expression plasmid vectors that encode growth hormone releasing hormone followed by electroporation. The result indicated that the intermuscular injection pM-GHRH plasmid mediated by electroporation on lamb and pig growth does not have the remarkable influence.
Objective: To construct the BD bait vector in yeast two-hybrid system by using pBK-CMV-HA-EBP50 plasmid. Methods: Coding region was obtained from the EBP50 plasmid by PCR. EcoR I/ BamH I -digested PCR products and bait vector pGBKT7 were ligated, and then transformed into E.coli DH5α to obtain the transformants. The positive clones were screened and identified by restriction endonuclease analysis and DNA sequencing. The correct recombinant plasmid pGBKT7- EBP50 was transformed into the competent yeast AH109. The activation of pGBKT7- EBP50 on the reporter genes and its toxicity on AH109 were also determined. Results: The size of the inserted fragment and recombinant plasmid was consistent with theoretically predicted value. There were no activation of pGBKT7- EBP50 on the reporter genes and no toxicity on AH109. Conclusion: The bait plasmid pGBKT7- EBP50 is successfully constructed for the yeast two-hybrid system., may be used to screen for the novel protein-protein interactions and further contribute to the study of EBP50 roles in regulation of cellular functional activities.
The clenbuterol was conjugated with BSA by diazotization at a ratio of 17 to form CL-BSA complex. The conjugated molecule was injected into the Balb/c mice for hybridoma preparation. The results showed that the synthesized antigen could immune the mice successfully. The frequency of fusion and the positive cloning could reach 85.26% and 15.23%, respectively. Through four rounds of cloning, one optimal clone named 4H5 was obtained. The result of IELISA demonstrated that the 4H5 culture supernament was high specific activitie against CL and could reach to 1.265±0.060 (OD450nm), comparing to the ascites against BSA (OD450nm 0.060±0.006). The titer of ascites generated from 4H5 injection could reach to 3.2×106 with low affinity toward BSA, and the subtype is IgG1 with light chain κ.
Objective To prepare cisplatin polybutylcyanoacrylate stleath nanoparticle (CDDP-PBCA-mPEG ) and observe the different tissue distributions of the nanoparticles in the mice body after the injection via lateral tail vein , and to study the targeting distribution of CDDP-PBCA-mPEG on normal mice livers. Methods CDDP-PBCA-mPEG was prepared by double emulsion method，with the surface modified by methoxypolyethyleneglycol.High-performance liquid chromatography (HPLC) method was established using methyl alcohol and water(55 ：45) as mobile phase；detection was done at 210nm.Sixty mice were randomly divided into 2 groups with 30 mice in each group:non conjugated free CDDP group and CDDP-PBCA-mPEG group, A single dose of either conjugated or free cisplatin equaled 5mg / kg of body weight was delivered via the tail vein. Five mice in each trail were sacrificed at 15 , 30 minutes, 1 ,2，6 and 12 hours after the injection , respectively. The cisplatin concentrations in the collected livers, kidneys, spleens, hearts, lungs and plasma were demonstrated using a high performance liquid chromatography with fluorescence detector. Results The mean diameter of the prepared CDDP-PBCA-mPEG was 170.9 nm；the mean entrapment efficiency of the particles was 60.1% ；and the drug loading rate was 3.88%．The cisplatin concentrations of the mice liver in the experimental groups was significantly higher than those in the control groups( P < 0. 01 ) .The nanoparticle conjugated cisplatin was significantly lower than those in the control groups( P < 0. 01 ) and cleaned up quickly from the kidney tissues. Cisplatin was released slowly in the liver and plasma during the detection period in the experimental groups. Conclusion CDDP-PBCA-mPEG has the liver targeting with slow medicine release. It also decreases the medicine distribution in the kidney and other organs. In the treatment of liver cancer, the polybutylcyanoacrylate stleath nanoparticle system has a good liver targeting ability, which increases the anticancer activity and markedly decreases the toxicity of cisplatin.
The genomic DNA of the seaweed Kappaphycus alvarezii was extracted by CTAB method. The template, Mg2+, dNTPs, primer, Taq DNA polymerase concentrations, and annealing temperature and the best cycle times were optimized for ISSR - PCR reaction system in K. alvarezii with single-factor and orthogonal design experiment. In 20?L ISSR-PCR reaction system of K. alvarezii, the most suitable concentration of template, Mg2+、dNTPs、primer、Taq DNA polymerase were 15ng, 2.7 mmol/L, 0.2mmol/L, 0.1mmol/L and 1.5U, respectively. And the PCR program for ISSR was 5 min initial denaturation at 94℃, then followed by 32 cycles of 50 seconds at 94℃ (denaturation), 60 seconds at 48℃ (annealing), 120 seconds at 72℃ (extension), and a final 8 minutes extension at 72℃. These results consequently provide a useful means for the research of genetic diversity, molecular marker, germplasm identification and auxiliary breeding in Kappaphycus and Eucheuma
Abstract: To overcome the difficult of extraction RNA from Euonymus japonicus 'Cu zhi' tissue with high levels of phenolic compounds and carbohydrates, the study modified the SDS/phenol method, and isolated total RNA from the leaf and stem tissues of Euonymus japonicus 'Cu zhi'. The results showed that, the lightness of 28S rRNA in extracted total RNA was two times as much as that of 18S rRNA, the A260/A280 ration is 1.94, and the A260/A230 is 2.06.The yield of RNA was 252μg/g (gram fresh weight).The p5cs gene fragment(EU347715) was successfully amplified by RT-PCR suggested that, the total RNA extracted was of high integrality, high quality and high productivity. The total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.
An improved SDS method was used to extract genomic DNA from Streptomyces gilvosporeus. The RAPD conditions were optimized by single factor experiment and orthogonal experiment, including Taq DNA polymerase, Mg2+, primer , dNTPs and template DNA. The results were as follows: DNA 60-150 ng, Taq DNA polymerase 1.0-1.5U, primer concentration 0.3-0.4 mmol/L, dNTPs concentration 200-250 μmol/L, Mg2+ concentration 2.5-3.0 mmol/L in 20μL PCR system. Under above optimal conditions,the abundant, stable and clear strips could be obtained.
The inclined gravitational settler showed some advantages in animal cells perfusion culture process. However, the further application of this settler had been limited because the cells were easy to deposit on its bottom. The properties of three-dimensional flow field inside the settler, which would influence the cells deposition, was investigated through the methods of combination of CFD technology and cold model experiments. The result showed that the amount of cells hung in the boundary layer were larger than that of adhered on the bottom. One of the alternative methods for this problem was to optimize settler structure by reducing the side-effect of boundary layer. With the result demonstrated that the amount of cells deposition could be reduced effectively in this optimized settler.
The serial analysis of gene expression (SAGE) technology is a very useful skill to do the researches on gene. It has many advantages, such as high throughout, high-fidelity, qualitation and quantitation. Based on our own researches and combined with the reviews of the related literature at home and abroad, we analyzed and evaluated the latest development of the research on SAGE, especially the development of principles, methods and applications of the technology. The improvements include three aspects, the optimization of SAGE itself, analysis among several SAGE libraries, combining with other gene researching methods. Through the whole view of SAGE used in liver researches, we find that the SAGE technology has been improved to some degree, especially, it's applications in many fields are greatly extended. The principles of SAGE change little, the methods of SAGE has been improved a lot.
In vitro culture of spermatogonial stem cells is a basic technology of biology ,which can promote the biology investigation of spermatogonial stem cells . So spermatogonial stem cells in vitro culture has become one of the hot fields in the Biology of the Stem Cell in recent years .Obtaining SSCs with high purity , plenty of vigour ,biological safty are the essential steps for SSCs related biological studies in vitro . However ,there are many factors affecting SSCs in vitro culture , such as animal species and age , culture medium , serum , selection of feeder layer and growth factors . SSCs always be thought not to culture in a long-term time in vitro ,which retain stem cell activity . But with the deeper and deeper investigation , people have already obtained the long-time culture of SSCs , specially in Murine . Murine is a direct investigation analogue organism to research mammalian even human in the end .This review mainly summarize the factors ,which affect SSCs in a long-term culture in vitro according to the recent related reports and our experimental work
Amniotic epithelial cells (hAECs) and amniotic mesenchymal cells (hAMCs) derived from humam amniotic mambrane express stem cell markers and have the ability to differentiate toward all three germ layers. Therefore, human amniotic membrane cells draw great interest as a source of for regenerative medicine. Recent reports indicate that hAECs or hAMCs possess the potential of multilineage differentiation, and can differentiate into "neuron-like" cells, "hepatocyte-like" cells, "cardiomyocyte-like" cells, pancreatic beta-cells, osteoblasts, chondrocytes and so on in vitro condition. The therapy-based expremental studies show that hAECs and hAMCs have the protective effects on neurological disorders, liver disease, myocardial infarction and diabetes mellitus. Recent advances in phenotypic characteristic and phenotypic plasticity of human amnion-derived cells were reviewed.
Highly unsaturated fatty acids（HUFAs）play important physiological roles in biological organisms. Human being and animals, including most of marine fishes, depend on dietary HUFAs because of deficiency in the activity of one or more of the key fatty acid desaturases required for HUFAs synthesis. So far the desaturase study is significant in science and application. The present paper reviews the recent progress in the studies of molecular biology of the desaturases of HUFA biosynthesis, emphasis on the cloning, expression and function analysis of the desaturase-gene.
Highly unsaturated fatty acids（HUFAs），especially ω-3HUFAs, are essential nutricients and play many physiological roles in human being and animals, including hydro-animal. However, the organisms depend on dietary HUFAs because of deficiency in the activity of one or more of the key fatty acid desaturases required for HUFAs synthesis, resulting in the desaturase study to be one of the heatst fields at the moment. The present paper reviews the recent progress in the studies of molecular biology of the desaturases of HUFA biosynthesis, emphasis on the structure and fuction of the desaturases.
Cell culture on microcarrier is a new kind of large scale culturing technique, and which is used by many bioproducts, such as vaccine and antibody. In this paper we introduce the history of cell culture briefly, and discuss the selection of the microcarrier and bioreator, described the mode of cultrue, the enviroment in the bioreator, and the prospects of this technique.
Spermatogonial stem cells (SSCs)，the only stem cells that are capable of transmitting genetic information to subsequent generations in adult animals，have abilities to self-renew and differentiate into spermatozoa. Therefore, SSCs are not only the study object of stem cell biology, but also the valuable resource of in vitro spermatogenesis, gene analysis and functional genomics. The present paper reported the study history and recent progress of in vitro culture for SSCs, and summarized the influence factors of SSC in vitro culture and maintenance mechanisms of SSC self-renewal.
Abstract: In stem cell research, more and more attentions were paid on the SP cells, now SP cells were detected in a variety of biological tissues, and they were similar with the stem cells of the same tissue in function and molecular markers on the surface. Especially in research of tumor stem cells, recently it was found that SP cells had the characteristics of tumor stem cells. They were isolated from tumor cells, and cultured ,which can be used to study some characteristics of tumor stem cells.But more reseachs need be done for SP cells.
It is of primary importance to construct a suitable gene delivery system in the technology of transgenic animal generation and gene therapy, which can effectively mediate the exogenous genes expressed in the target cells. Vectors used for the genes transfer can be classified into virus vectors and non-virus vectors, thereinto, researches on virus vectors trend to be a hot spot in the technology of gene engineering attributing to its high efficiency of transfection and the stably expression of exogenous genes . However, because of the diversity of virus category, specificity of its structure and its complicated parasitic relationship with host organism , scientists are still working on being more familiar with its functional mechanism，life cycle and relationship with pathogenesis, which makes kinds of virus still not yet to be developed as gene delivery vector. Presently, just a few kinds of virus have been successfully reconstructed as gene delivery system, and its progress in transgenic technology will be summarized in this essay.
Hyaluronic acid (HA) is a high molecular weight polysaccharide . Because of its special molecular structure, physical and chemic character, and physiological role, it has broad application in cosmetic industry, corrective surgery, healthcare products, medical research, clinical therapy, and tissue engineering. In this paper, we discuss the presently advance in application and preparation of HA at home and abroad.
Foot-and-mouth disease(FMD) emergency vaccines was a high potency vaccine (usually ≥6 protective dose 50 (PD50)) ,which had wide cross-immunity within FMD serotypes, and could induce rapid protective immunity after animals were inoculated 3～4 days, moreover, used emergency vaccine could prevent or decrease local virus replicating in throat and other sites, where is adapt to FMDV existing, so dramatically reduced the amount of virus released into environment and occurrenced of persistently infected "carrier" animals following vaccination, furthermore, the antibody induced by emergency vaccines maintaining longer than traditional inactived vaccines. The succeed using of emergency vaccines by Netherlands and Greece in 2001 and 90 times, respectively, indicated that emergency vaccines was advantage of defending emergency conditions. Lately, several studies carried out by world foot-and-mouth disease referenced laboratory and other countries approving that emergency vaccines had unexampled preponderance whether in preventing direct infection or indirect infection .At present, emergency vaccines were considered by UK and other free Foot-and-mouth disease countries as an additional means for control. This article described emergency vaccines from its antigenic coverage、immunity potency and rapid immunity mechanism.
The 3C Protease from foot-and-mouth disease is critical for viral pathogenesis,having vital roles in both the processing of the polyprotein precursor and RNA replication.Although recent structural and functional studies have revealed new insights into the mechanism and function of the enzyme,key questions remain that must be addressed before the potential of FMDV 3C as an antiviral drug target can be tealised.
Secretory phospholipase A2 (sPLA2) is an important component of snake venom which has a variety of biological activities. They exert their different functions by molecular and structural diversity, eventually triggering kinds of physiological and patho-physiological effects. This review summarized the structure and biological activities of snake venom sPLA2 with emphasizing its relationship between diverse pharmacological effects and structures. In addition, some mechanisms of sPLA2 actions and hot research field of sPLA2 were also discussed.
Abstract: Disulfide bonds play an important role in stability of protein conformation and maintaining of protein activity. However, formation of natural disulfide bonds is a rate-limiting step in protein correct folding. With study on protein disulfide isomerase and folding intermediates, folding mechanism of disulfide-rich proteins has been clarified gradually. The protein folding mechanism is focused on in this review, which was understood by formation of disulfide bonds in vivo and protein oxidative folding in vitro, and its application in genetic engineering. These new findings may represent future directions for improving the quality of recombinant protein which is rich with disulfide bonds
Solution flow is inevitable inside a protein solution during protein crystal growth process. In this paper, the proceeding of the effect of existence of solution flow, including forced solution flow, stirring, and crystal growth on a rotary plate, on protein crystal growth have been reviewed. Moreover, together with our primary experimental results, the improvements of crystal growth on a rotary plate have been emphasized in this paper.
This paper introduce the DNA techniques for identification of animal Fibres and discuss using DNA to identification of Animal fibres is Important for cashmere and wool industry.Up to date, the progress of the techniques is in home and overseas.
Defensin is a kind of polypeptide that has specific immunity against bacteria, fungi, virus and other pathogenic microorganisms. It possesses specific characters, such as heat- durability, broad - spectrum antibiotic and special antimicrobial activity. With the further research progress on defensin, it will have a tremendous impact on medicine field and play an important role on plant genetic engineering and variety improvement of plant. So far, defensin had been transformed to varieties of plants and expressed in this plants.It makes an important action on plant genetic engineering against pest and disease. In this article we briefly review the classification of different defensin, their specific immunity against bacteria,their biological functions and progress in plant genetic engineering.In the end we make the forecast to its prospect of application.
The study of plant protoplast separate, culure, fusion and regeneration. According to literature analysis, it discusses the question of theprotoplast wok and the tendency of development in the future. Simultaneously, it indicates the rapid development with liberation, culture and fusion of plant protoplast in 20 years.
Positive selection marker had been widely accepted by people as selective gene for unique advantage compared with negative selection marker and gradually used in plant genetic transformation. The principle and the applications in recent years of positive selection were described in this article.
Most plant fragrance compounds belong to three major groups: phenylpropanoids (including benzenoids), fatty acid derivatives and terpenoids. Their pathways with respect to the enzymes and genes involved and the underlying molecular mechanisms controlling them were elucidated.The research and application in molecular biology were introduced. All these open up theorectic guidances for the generation or manipulation of fragrance compounds.
As a prefatory topic in genetic engineering technology, the construction of transgenic bacteria has the good industry prospect. Construction includes its best expression vector construction, the choice of suitable host cells, transforming, the identification and screening of genetic stability, the genetic stability of the plasmid, and high-level expression of the transgenic bacteria. This article reviews the transgenic engineering bacteria and vector about the construction, the acceptor bacteria such as Escherichia coli, lactic acid bacteria, yeasts and cyanobacteria selection and breeding, exogenous gene expression and application in aspect on agriculture of genetic engineering feed phytase bacteria, pesticides and pesticide Rhizobium microbial genetic engineering, and pointed out the safety problem and the imperfect aspect of transgenic bacteria in the application.
L-phenylalanine (L-Phe) is one of essential amino acids for humans. It is an important chemical intermediate in medicine and food industry. There is a huge market for L-Phe. The demand of L-Phe in all over the world reach 30000 ton in 2005. Especially, phenylalanine ammonia lyase (PAL) catalyzing trans-cinnamic acid (t-CA) to L-Phe method become hotspot in the production of L-Phe due to great demand for artificial sweetener aspartame and the descrease of the cost of tran-cinnamic acids. In the paper, we summarize the progress in the production of L-phenylalanine by using PAL.
It introduces the physico-chemical chemical and physical propertiyes of itaconic acid. There are Ttwo main production methods to produceof itaconic acid，which are , chemical and biological fermentation. Many researches have been focus on choices of raw materials and strains, application of deep fermentation and immobilization technology in fermentation process, comparison of re-crystallization ,ion exchange resins , organic solvent in the extraction process in the microbiological fermentation.Focused on the method of microbial fermentation introducing. Research on raw materials, fermentation bacteria choice, fermentation process such as deep fermentation, immobilization technology. The comparison of three extraction technology, such as re-crystallization process, ion resins exchange technology, organic solvent extraction method. The production status of itaconic acid and production capacity of all componies in domestic and international market, their is production capacity are overviewed. Besides, the products of itaconic acid and its derivatives products in the new highly efficiency efficient deodorant, cleaning industry, adhesives and other industrial applications araree detailed summarized. The author also estimates the market application prospects of itaconic acid. was also forecasted.
As one of biomass energies, fuel-ethanol could serve as a new energy for solving global warming and fossil fuels shortage. Research status of lignocelluloses pretreatment, saccharification, and ethanol fermentation is detailedly introduced, and then research progress of biomass-generated syngas to fuel-ethanol is also reviewed. At last, prospect and development direction of fuel-ethanol from lignocelluloses is expected.
The essential of TCM modernization is how the modern technique, scientific theory and culture are used in TCM research. This article reviewed that biotransformation technique can elicit new ideas and approaches in TCM resource exploiture or new effective compounds discovery, and also can promote the modern research of TCM pharmacy and pharmacology.
Biotechnology is one of the five strategic emphases in the next 15 years in China. As one of the most important application fields of biotechnology, pharmaceutical biotechnology is its front technique and the hotspot of its R&D, as well as the most important technical impetus for the development of pharmaceutical industry. This article analyzed and summarized the strategic opportunities and six challenges of Chinese pharmaceutical biotechnology and its industry. The strategic opportunities included building an innovative nation, constructing a harmonious society, and adjusting the economic structure. The challenges included lack of innovative abilities, smallness of industry size, insufficient of capital investment, incorrectness of R&D main body, low of patent quality and IPR consciousness, and faultiness of standard system.
industry-universty collaboration(IUC) is one of the most important ways to improve the science and technology innovation ability of our domestic biomedicine industry. The problems and barriers of IUC of biomedicine industry in China are unique. According to a reasearch about some universities,institutes and biomedicine companies, we put forward some problems about IUC in science and technology innovation of biomedicine industry, and make some suggestion about it.
Pharmaceutical biotechnology is the most important application field of biotechnology, its front technique and the hotspot of its R&D. We had a narrow gap with developed countries in the field of pharmaceutical biotechnology, and had the opportunity to catch up with them within a short term period. Speeding up the development of pharmaceutical biotechnology and its industry has profound strategic influence for transformation of economic growth mode and raising people's living standard. This article analyzed and summarized the strategic aims, emphases and guarantee measures of Chinese pharmaceutical biotechnology. It delivered four strategies and seven measures to boost the development of Chinese pharmaceutical biotechnology.
Proteomics study developments rapidly in China, which is becoming more important in the world. Adopting bibliometrics, this paper shows some development suggestion of proteomics in China, including innovation in basic study, development system dominated by government etc.
