Cross-linked Enzyme Aggregates is a novel carrier-free immobilized enzyme. The preparation consists of two steps :physical aggregates and chemical cross-linking .This immobilizing technique made enzymes more stable in extreme environments. The following aspects were described in detail :the preparation ,the property and the advances of CLEAs.
The principal purpose of this study is to set up the isolation methods for diphtheria toxin expressed by genes, to purify the toxin with complete biological activities, and to make researches on its toxicities. In the experiments, the target gene was inserted into the cloning vectors--pET22b, then the vectors were introduced into host cells -BL21(DE3). The expression of the toxin was induced by addition of IPTG. The expression products were purified by His-6 label affinity chromatography. Then the diphtheria toxin expressed was transferred to PVC membrane using semidry blotter to determine the amino acid sequence of N-terminal. The "nicked" diphtheria toxin linked by disulfide bond was gained by protease hydrolysis. At the same time, acute toxicities of diphtheria toxin in guinea pig, cytotoxicity in three kinds of cells were tested. The results indicated that the expression system established in the study expressed DT effectively. Amino acid sequencing of expressed DT gave the N- terminal sequence- MGADDVV…. This was identical with the experiment project. The LD50 of DT in guinea pig (ig) was 0.64μg/kg; and the IC50 values of DT in CHO-K1, BS-C1 and Hela were 1.709μg/ml, 1.424ng/ml and 28.947ng/ml respectively.
Six set primers for influenza A virus polymerase gene PB2、PB1 and PA were designed. With the primers, PB2、PB1 and PA gene were amplified in two parts. Because the primers contain the Ⅱs restriction enzyme cleavage site at 5' end, the amplified products could be cloned into pHW2000 orientionationally post digestion of these fragments with restriction enzymes BsmBⅠ、BsaⅠ or AarⅠ. By detecting the expression of the report gene-enhanced green fluorescent protein (EGFP), the polymerase activity of recombinant plasmids can be proved directly. The cloning strategy was proved to be simple、quick and easy to construct the expression plasmids of influenza A virus polymerases gene for reverse genetics systems, and make latter work easy.
Adiponectin, as a specific adipokine, entails diverse biological roles associated with obesity, insulin resistance, typeⅡdiabetes and cardiovascular diseases, with broad potentials of application. In this study, by using the total human blood genomic DNA as template, we particularly exploited the approach of splicing by overlap extension (SOE) to combine the two individually amplified exon fragments containing adiponectin-coding regions, and obtained the accurate, full-length of adiponectin gene (AP) verified by sequencing analysis. Subsequently, an expression construct pET-wAP was achieved by fusing the adiponectin gene behind the thioredoxin region within the potent plasmid pET32a(+). The fusion form of thioredoxin-adiponectin was successfully expressed by IPTG induction, with a high yield up to 41% of the total cell protein but mainly in inclusion bodies. Further studies on efficient and soluble expression of human adiponectin and its globular domain are now in proceeding.
Objective To produce the SARS immunoglobulin for intravenous injection from convalescent plasma. Methods Combination of the protein purification process of cold ethanol with ion exchange chromatography.Treat the source plasma with S/D，incubate the bulk at low pH and filtrate the bulk with nanofilter. Results The purity of the product was 99.0%,the monomer and dimmer contents of IgG was 100%,the ACA was 11%,the PKA wasn’t detected,the sterlity,safety,pyrogen and stability tests met the Chinese Requirements of Biological Products. the product was specific to SARS antigen and the titers of the product was1:83(ELISA),1:1600(IFA) and 1:200(NT)。Conclusion The process was feasible, the product had high titer SARS-antibody and the quality of product met the standard of Intravenous Injection.
A bacterial phage f105 based recombinant Bacillus subtilis for secretive production of penicillin G acylase (PGA) was constructed and expression of the cloned gene was optimized. Both the promoterless penicillin G acylase gene (pga) and the native promoter bearing penicillin G acylase gene (pgaPB) were isolated by PCR amplification from the chromosomal DNA of Bacillus megaterium with properly designed primers. The isolated genes were then inserted into the chromosomal DNA of a lysogenic B. subtilis bearing prophage f105MU331 through homologous recombination, with the aid of the integrating vector pSG703 that contained the homologous fragments of the prophage f105MU331. The resultant recombinant strains were denoted as B. subtilis PA1 and B. subtilis PA2, respectively, in which the recombinant genes were covalently integrated into the chromosomal DNA, and was located downstream of a strong thermal inducible phage promoter. The recombinant strains showed high stability and effective recombinant PGA production. The productivity of recombinant PGA from B. subtilis PA2, in which transcription of the heterologous gene was initiated from the phage promoter and the native pga promoter, was higher than B. subtilis PA1 where transcription of the pga gene was initiated from the phage promoter only. Inducing the recombinant strain at 50 ℃ for 3 min in the late exponential phase of growth led to best PGA production. Addition of phenylacetic acid failed to stimulate PGA production, and glucose exhibited inhibitory effect on PGA production. The pH stat fed-batch fermentation with strain B. subtilis PA2 was carried out on a 20 L fermentor, and a high productivity of 18.28 U/ml was achieved.
High-voltage electrostatic system was used to prepare alginate-chitosan microcapsules for Immunoglobulin of Yolk (IgY) via electrostatics interaction. Optimum conditions were established for preparation of homogenous, spherical, and smooth alginate-chitosan microcapsules. The entrapment efficiency , in vitro release of the microcapsules in SGF (Simulated Gastric Fluid) and SIF (Simulated Intestinal Fluid) and IgY activity retained were examined. IgY entrapment efficiency achieved 90% in the optimum condition. The IgY retained 80% activity after 2 h exposure to SGF and then 6 h exposure to SIF. The microcapsules released less than 10% IgY upon exposure to SGF for 2 hours, and released more than 90% IgY upon exposure to SIF for 6 hours. Electrophoretic studies of IgY microcapsules passed gastrointestinal tract showed the intact protein band. Consequently, it can be said that the system can be offered for the temporary protection of IgY against acidic and enzymatic degradation during gastric passage, enabling its use for oral passive immunotherapy.
Abstract: This study was designed to investigate the usefulness of urinary nucleosides in the diagnosis of primary hepatic carcinoma. By using micellar electrokinetic capillary chromatography (MECC )method , urinary nucleosides were determined in an uncoated 50cm×75μm i. d capillary tube using a optimized 250 mmol•L-1SDS–20 mmol•L-1 sodium tetraborate–40 mmol•L-1 sodium dihydrogenphosphate buffer（pH=6.8）, 10kV voltage, 2psi×s injection, 25℃ temperature and UV detection at 260nm. The nucleosides were extracted from urine by phenylboronate affinity gel chromatography. Ten kinds of normal and modified nucleosides were determined in urine samples from 20 healthy individuals and 20 patients of primary hepatic carcinoma, it was showed that the contents of urinary nucleosides excretion, especially modified nucleoside in patients of primary hepatic carcinoma were higher than those in normal healthy persons. These results provided a kind of probably means for the assessindividualst of the urinary nucleosides in cancer clinical diagnoses.
Inducible heat shock protein 70 (HSP70) is a major heat shock protein induced under stress conditions and functions mainly as chaperone. HSP70 molecule consists of two domains, N-terminal ATP binding domain(ATPBD) and C-terminal polypeptide binding domains(PBD). A nuclear localization signal(NLS) is within ATP binding domain(ATPBD). The DNA-coding regions of full-length HSP70(HSP70wt), HSP70ΔATPBD, HSP70ΔPBD and HSP70ΔNLS were cloned into a bacterial expression vector pQE-31, respectively, by PCR. HSP70 and its truncated mutants were over-expressed in Escherichia coli M15 and the fusion proteins were purified with Ni2+chelating affinity column and further purified by DEAE-cellulose to apparent homogeneity. Based on SDS-PAGE, both HSP70 and HSP70ΔNLS were 70 kDa, HSP70ΔPPBD 52 kDa and HSP70ΔATPBD 28 kDa. HSP70, HSPΔATPBD, HSP70ΔPPBD and HSP70ΔNLS that had been electrophoresized on SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (PVDF) were all recognized on Western immunoblot by anti-human HSP70 antibody. In PBS buffer, HSP70wt, HSPΔATPBD, HSP70ΔPPBD and HSP70ΔNLS were all proved to exist in monomers using gel filtration chromatography. In order to check whether the recombinant proteins can interact with nucleolin (C23) , a protein dominantly distributed in nucleolus. The purified proteins that had been fractioned on SDS-PAGE and transferred to PVDF were first incubated with nuclear extracts containing nucleolin and then visualized by anti- nucleolin antibody (protein overlay assay). The in vitro protein overlay assay showed that HSP70 and HSP70ΔNLS and HSPΔATPBD can interact with nucleolin, while HSP70ΔPPBD can not, suggesting that the C-terminal polypeptide binding domain of HSP70 is sufficient for the interaction between HSP70 and nucleolin.
The liquid fermentative conditions of Enterococcus sp. were studied through single factor and orthogonal design experiment. The best medium for L-α-Glycerophosphate oxidase(GPO) formation was composed of glucose 0.1%, proteose-peptone 1%, yeast extract 1%, K2HPO4 0.5%, glycerol 0.2%, Salt solution 0.25 ml , the initial pH was 6.8 .Furthermore, some research about the conditions of fermentation was done. When the germ grown in 250 ml flask containing 100 ml of the medium on the rotary shaker (200r/min) at 30℃ for 18h , GPO had maximal activity, which was 33.8 times than original production, During the course of scale-up culture, keeping other culture conditions, culture circle was shorter 6h than the one of flask fermentation, and the former enzyme activity increased 61% than the later, when the fermentation speed was 100r/min and dissolved oxygen was 6L/min. In addition, optimum temperature and pH of the enzyme action were 37℃ and 7.5~8, respectively. When GPO was treated with in the range of pH9.0～9.5 at 37℃ for 1h,the enzyme activity was kept only 33%.
Human (homo sapiens) insulin-like growth factor-1 (hIGF-1) gene coding for the mature protein was synthesized and cloned in a pMD18-T vector, and then transferred into prokaryotic fusion expression vector pGEX-2T. This fused hIGF-1-GST gene was under the control of tac promoter and the fusion expression of hIGF-1 with an Glutathione S-transferase (GST) tag in the E. coli BL21(DE3) upon induction with isopropyl-1-thio-beta-D-galactoside (IPTG). The recombinant protein showed a molecular weight of about 35 kD and was mainly in the form of inclusion bodies according to SDS-PAGE. The insoluble hIGF-1-GST was solublized from inclusion bodies by denaturation using 6 M urea in the presence of DTT. Both the recombinant hIGF-1-GST from the soluble fraction and the dissolved inclusion bodies of the crude extract of culture could be purified to over 90% purity by using GST-tag affinity chromatography. The purified hIGF-1-GST was again confirmed by SDS-PAGE and Western blot analysis. The expressed protein contained within the inclusion bodies was refolded and renatured to natural structure. After cleavage of the soluble and renatured fusion protein with thrombin, the released hIGF-1 was respectively purified by GST-tag affinity chromatography again and prepared for MTT colorimetric assays. Both the two hIGF-1 preparations could promote NIH/3T3 cells proliferation in a dose-dependent manner. The bioactivity of the soluble hIGF-1 was as potent as that of the standard hIGF-1 in cell culture bioassay
Abstract : callus were induced from cephalotaxue fortunei Hook.f, the effect of hormone, sugar concentration, temperature and quantity of inoculation on the inductivity were studied. under the condition of 3% sugar concentration, 0.75% agar concentration, and 25±1℃,the rate of callus inductivity reached to 90%.the callus inoculation of 2.0g accelerate the growth of callus in a flask 100ml.
Rice cyclin-dependent kinase inhibitor(ICK)is a key control protein in cell-cycle, it can effect the growth and development of cell. Rice OsICK1 gene was expressed in BL21,with the prokaryotic expression vector pET-28a(+).And anti- OsICK1 polyclonal antibody was obtained by injecting OsICK1 into mouse. The result of Western blotting show that the polyclonal antibody not only can differentially combine with the expressed protein OsICK1, but also can differentially combine with the OsICK1 in rice.It have established the base for the research of OsICK1 gene expression in rice.
In order to study gene express related to flower development of Lycoris longituba，total RNA from the petals of Lycoris longituba was extracted and purified by using the modified acid guanidinium thiocyanale-phenol-chloroform extraction method and the mRNA was isolated from total RNA by using Biotin-labeled Oligo(dT) and SAPMPs. The cDNA was synthesized and catalysed by reverse transcripase. The cDNA was then linked with EcoRⅠAdaptor. And the ends were phosphorylated. The Xho Ⅰdigestionreleased EcoRⅠAdaptor. The fragments smaller than 500 bp were removed by gel-purification and ligated to vector to transform the host by electroporator。 Finally the cDNA library of Lycoris longituba was constructed. The sink size of the primary cDNA is 1.112×106, in which 96.8% phages were recombinant, insert sizes ranging from 0.8～3.0kb were obtained. All of the above mentioned accorded with the general requirements of cDNA library construction, which established a good foundation for investigating the flower development of Lycoris longituba and cloning full length DNA of related genes with the methodology of both genomics and molecular biology.
Objective To establish a quick and simple method used for evaluating capability of sperm cells up-take exogenous DNA. Methods After co-cultured with digoxigenin (DIG) labeled DNA, frequencies of sperm cells carrying foreign DNA were tested by immunocytochemistry and compared with corresponding results from liquid scintillation counting. Results Sperm cells carrying exogenous DNA were dyed heavily red and control sperm cells did not. At the same time, the process and location of exogenous DNA interacted with sperm cells were also displayed. Furthermore, results came from immunocytochemistry had obviously relationship with that that from liquid scintillation counting, a classical method to evaluate sperm capability to take up foreign DNA (r = 0.96). Conclusions Imunocytochemistry is not only a safe, quickly and sensitive method in estimating the capability of sperm cells to take up exogenous DNA, but more information about interaction between sperm cells and exogenous DNA could also be revealed.
The coat protein gene of peanut stripe virus (PStV-cp gene) was obtained from pGEM-StV digested by BamHI and SpeI, and plant expression vector pBI-StV was constructed by inserting PStV-cp gene fragment into pBI121 between BamHI and XbaI sites. The vector pBI-StV was characterized with PCR screening、restriction endonuclease mapping.
Abstract: we cloned two completely new MADS-box genes from Castanea mollissima by the method of 3’RACE, named CmMADS2(DQ148294) and CmMADS4(DQ148296) respectively. CmMADS2 encoded 242 identical amino acids (aa) including 56 aa of the MADS-box domain and CmMADS4 encoded 242 identical amino acids (aa) including 72 aa of the MADS-box domain. RT-PCR analysis showed that CmMADS2 expressed in inflorescences and tender leaves with flowers, not in other tissue(fruit﹑old leaves﹑stem and tender leaves with no flowers). While CmMADS4 was expressed in all the tissue mentioned above, suggesting that these novel MADS-box genes may be involved in floral organ development of Castanea mollissima.
The sensitivity, specificity and application and expenses among the gold immunochromatography assay (CGIA), DIA (Dot Immunobinding Assay) and PCR assay have been compared. Use trisodium citric acid deoxidization to prepare colloidal gold, and then it was coupled with rabbit polyclonal antibody of Xac04 pathogen, to produce the fast reagent strip of CGIA. A new useful method for rapid screening assay of CBCD in field was developed.
The objective of this study was to develop a practical in vitro culture system of chicken fibroblasts to facilitate further studies on the feeder cells of chicken embryonic stem cells. Explant techniques and monolayer culture in culturing chicken fibroblasts were compared, the primary chichen fibroblasts derived from tissue explant culture grown slowly and confluented together by 6～7 days of culture, while cells derived from enzymatic digestion grown faster and confluented together by 5 days of culture. However the growing speed of passaged fibrlblasts derived from the two methods was similar, which could confluent within 2 to 3 days of culture. Chicken embryonic tissue digested by 0.25% trypsin provided a high yield of viable proliferating for primary culture. Homogeneous fibroblasts could be obtained after the mixture of fibroblasts and epithelial cells were treated by the combined use of typsin digestion and repeated attachment method for three or four subcultures. There were 75%-80% of cells survived after frozen and thawed. The growth curves of fetal and skin fibroblasts were normal and similar. Chicken embryonic and skin fibroblasts could subculture 12th generation and still retained the normal chromosome content.
The metabolites of endophytic fungus No.1028 which was isolated from the stem of Gingko had strong inhibition effect on Alternaria solani、Fusarium oxysporium、Colletotrichurm capsici、Colletotrichum gloeosporioides、Thanatephorus cucumeris and Rhizoctonia Solani . The optimum medium composition and fermentation condition of No.1028 were studied. Through orthogonal test, the result showed that the optimum medium was glucose 3.0%, corn starch 1.0%, soybean meal 2.5%, NaNO30.5%, KH2PO40.4%, FeSO40.5mg/L, CaCO30.5%. The optimum fermentation condition was culture temperature 28℃, initial pH7.0, culture time 72h, medium volumes 100mL/300mL.
Insect defensins as a sort of defesins are a family of novel peptides with anti-extrogenous pathogen activities. They are a class of small cationic antibiotic peptides that have abundant of arginine (Arg). Insect defensins selectively kill Gram-positive bacteria. Using the preferential condon of Pichia pastoris, a novel antibacterial peptide Musca domestica Defensin (des-hf) 140bp in length was designed and synthesized according to the sequence from GenBank（AY260152）. The modified antibacterial peptide gene was cloned into the pPICZα-A vector to construct the recombinant expression vector pPICZα-A-des-hf and then transformed into yeast host strain SMD1168. Under the control of the promoter AOX1 (alcohol oxidase 1), the protein with a similar molecular weight to native antibacterial peptide was expressed and secreted into the supernatant. Des-hf peptide showed potent antibacterial activity against Staphylococcus aureus in elementary Secretion expression.
Xylanase gene xynB of Aspergillus sulphureus was amplified by RT-PCR. Sequence analysis showed that xynB composed of 678 nucleotides (nt), which encoded a 24 kDa protein, shared more than 95% similarity to the xynB sequences of A.niger opened in GenBank. xynB was overexpressed in Escherichia coli BL21 induced by IPTG after it was ligated into vector pET28a(+). The recombinant enzyme was optimally active at 40℃ and pH4.4. The xylanase activity just decreased 13% after 6 h in pH2.4 buffer. It has no obvious influence on enzyme activity after incubation at 30~50℃ for 30 min. XynB enzyme retained higher residual activity when different concentrations of Cu2+, Zn2+ and Ca2+ was added to the catalytic system respectively.
Abstract：Based on the published nucleotide sequences of Influenza A virus, Porcine reproductive and respiratory syndrome virus, Porcine cirocovirus-2 and Porcine pseudorabies virus, 4 pairs of primers were designed. And conserved DNA (or cDNA) fragments of the 4 viruses obtained by using PCR (or RT-PCR), were ligated with pMD 18-T Simple Vector to construct recombinant plasmid for DNA probe preparation. The recovered DNA probes were spotted on located sites on nitrocellulose filter to prepare low density DNAarray. During the process of multiplex PCR, samples were labeled by Biotin-11-dUTP. Then the labeled PCR products were hybridized with DNAarrays, and the hybridization signals were scanned and analyzed by scanning device and software. The results showed that this DNAarray could identify and distinguish the 4 viruses in samples synchronously and was more sensitive than PCR method. This method can be use for lab diagnosis, large-scale epidemiology investigation and quarantine of animal and animal products.
Abstract: Objective: It is applied to canine STR genotyping by studying the optimum amplification condition of mutiplex PCR system consisted of STR loci PEZ1, PEZ5 and PEZ20. Methods: Gaining the best concentration of the three primer pairs by Orthogonal design, then optimizing the concentration of Mg2+ and dNTP. The amplified products were detected by polyacrylamide gel electrophoresis and silver strains. Results: In condition that the concentration of three primer pairs PEZ1、PEZ5、PEZ20 which was 0.1μM、0.2μM、0.1μM severally, Mg2+ 3.0 mM and dNTP 0.2 mM , the objective strips of three STR loci are clear and similar intensity in this mutiplex PCR system, with faint and little of non-purpose strips. The mulplex system can applied to the canine STR genotyping test.
The structure gene apxⅡA of ApxⅡtoxin of Actinobacillus pleuropneumoniae (APP) serotype 7 was expressed in E.coli BL-21(DE3) with prokaryotic expression vector pGEX-6p-1, the expressed product formed inclusion. The inclusion protein was washed 2-3 times with 50mmol/L Tris-HCl (PH8.0), 1mmol/L EDTA buffer contained 0.5% Triton X-100, then followed by washing one time with 1mol/L NaCl. After washing, the inclusion protein was denatuated with 6 mol/L guanidine hydrochloride. Then the solution was diluted and dialyzed against 20mmol/L Tris-HCl (pH8.3), 1mmol/L EDTA supplemented with GSH, GSSG and 0.5 mol/L L-Arginine. The solution was then concentrated by PEG 20 000 and dialyzed against 20mmol/L Tris-HCl (pH8.3), 1mmol/L EDTA. After concentration and dialysis, the rApxⅡA protein solution was purified with MicroSpin GST Purification Module kit (Amersam) according to the manufacturer's instructions. The hemolytic assay showed that the inclusion protein restored hemolytic activity after be treated by washing, denatuation, renaturation and purification, it can lysis 1% sheep erythrocyte suspension.
Using RT-PCR technique, the entire FMDV-3C gene segment was amplified from suckling mouse carcass and cloned into pGEM T easy vehicle to obtain the recombinant plasmid pT-3C, which was confirmed by nucleotide sequencing. EcoRI cleavage was used to construct a 3C protease fusion expression vehicle, and recombinants having the correct orientation were screened with a pair of primers Pr78/3C-A2. The prokaryotic expression vehicle pSOC-3 C constructed was subjected to restriction analysis and, under the induction of IPTG, could in E.coli BL21 (DES)plysS express the SOC-3C fusion protein that gave a 26 KD-sized target electrophoretic band, the latter exhibiting immunoreactivity on Western-blotting. These findings paved the way for further research in bacteriophage surface display of the 3C protease and development of novel vaccines and pharmaceuticals
Malate dehydrogenase (MDH), an essential enzyme in many pathways, has been widely distributed from a variety of sources. Multiple amino acid sequence alignments are used to study the phylogenetic relationship of the MDHs from different sources, and show that there are three kinds of MDHs. Using technologies of bioinformatics, conserved structure and domains are analyzed, including coenzyme NAD (or NADP) bonding domain, active site signature and substrate bonding domain. The relationship between mechanism of catalysis and sequences, structures of MDH are also investigated. The application of bioinformatics in the research of MDH to redesign by rational was put forward.
The preparations and the characteristics of the chitosan-sodium alginate microcapsules containing immobilized α-amlyases are described in this paper. The results indicated that ：（1） the ideal spherical morphologies and the narrow range distributions of two different kinds of microspheres of immobilized α-amlyases are obtained; (2) compared with the free enzymes, there are more higher thermal and PH stabilities, excellent in the repeated use of properties and no deterioration in longer storage in the two different kinds of immobilized α-amlyases; (3) the michaelis constant (Km) and the maximum rate of two different kinds of immobilizedα-amlyases are larger than those of frees.
The paper deals with the culture medium compositions and fermentation conditions of biosynthesis of carotenoids from rhodotorula RY-051 strain. Through the determination of growth curve and optimization of fermentation conditions, some main parameters are selected, which are as follows: initial pH 4.5, temperature 26℃and fermentation time 64h. The composition of carotenoids was mostly by detection of thin-layer chromatography (TLC) and HPLC. The cell of rhodotorula RY-051 and the content of carotenoids were 6.89g/100mL, 5.55mg/L.
Comparative studies of enzymatic synthesis of citrate in the presence and absence of ultrasound were carried out in organic media. The influence of ultrasound power，organic solvent , water content，pH value on enzymatic synthesis was investigated. The optimal pH value for lipase subject to ultrasound was similar to that of the control condition，and Km value decreased. It was shown that suitable ultrasound could enhance reaction rate of lipase catalyzed－esterification in organic solvent.
Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)(PHBHHx) is a new type of biodegradable material synthesized by microorganism. The PHBHHx material-machinability properties depend very much on its molecular weight. When lauric acid was used as sole carbon source, 90~110×104molecular weight of PHBHHx was obtained by Aeromonas hydrophila 4KA4. The PHBHHx with moleculr weight ranging from 25×104 to 110×104 could be obtained when glucose added to the media in 30L and 4000L fermentors. It was showed possible that glucose can be used to control molecular weight of PHBHHx bio-synthesized.
The full length of AtPCS1 gene was amplified by RT-PCR. After sequencing, the gene was cloned into the expression vector pET28a to contrast AtPCS1 expression plasmid pET28a-AtPCS1. The E.coli BL21 contained the expression plasmid was induced by 0.75mM IPTG and the protein was purified using SDS buffer. Mouse was immunized with the purified protein. The results of ELISA showed that the titer of the anti-serum was about 1:2000 and the western-blot analysis of the anti-serum with purified protein AtPCS1-His or the total protein of Arabidopsis thaliana show the polyclonal anti-AtPCS1-His serum has high activity and speciality.
As lignin is a major structural component of plant cells, modification in lignin content may have ecological implications. Taking different transgenic cottons as test materials by a pot experiment, the lignin contents, POD and PAL activities in different cotton tissues were studied in this thesis and the changes of lignin metabolism in transgenic cottons were indicated. The results showed that the lignin contents in different plant tissues of transgenic Bt cottons were higher than that in their controls at the degree from 2.09% to 9.45%, and the differences among treatments were not significant. But the lignin contents in different plant tissues of transgenic Bt+CpTI cottons were lower than that in their controls at the degree from 9.19% to 31.95%, and only the difference in roots between transgenic and non-transgenic treatments was significant. In comparing with conventional cottons, POD and PAL activities in transgenic cottons had some changes in certain varieties and tissues. To transgenic crops, unintended effects would be more uncertain and complex along with diversification of transgenic genes.
FD-3 is a new screened strain that can biodegrade dibenzothiophene (DBT), a model compound in biodesulfurization research work, called Agrobacterium radiobacter. It converts DBT to 2-hydrobiphenyl (2–HBP).The organosulfur in diesel can be removed by FD-3. It not reduces the surful’s content in oil, but can’t destroy the carbon skeleton. This study researched the desulfurization characteristics of FD-3 resting cell to diesel oil organosulfur, and choice the best process by orthogonal experiments.
DNA shuffling is a practical molecular breeding process for the rapid evolution of genes by in vitro recombination. It has extended its researches and applications to a wide areas, including the improvement of the structure and function of proteins, specificity and activity of enzymes, small molecular biochemical pharmaceuticals and drug metabolic enzymes, gene therapy vehicles and transgenes, laboratory animal models and so on. In addition, DNA shuffling is also used as an effective theoretical researching tool in molecular evolution. Here we gave reviews in molecular evolution, especially the technology of DNA shuffling and its main applications and development.
The newly-synthesized protein must fold into its proper three-dimensional structure to be useful to the cell, in many situations efficient folding of many newly synthesized proteins depends on assistance from molecular chaperones, which serve to prevent protein misfolding and degradation in the crowded environment of the cell. In this article, at first we discuss the concept and classification of molecular chaperones, next describe several new hypothesises about how protein folds, and lastly use Hsp70 and Hsp90 as examples introducing the latest research progress on the function of molecular chaperones in facilitating protein folding.
The research of genomics has been transferred from the structural genomics focusing on determining the complete sequences of the genome to the functional genomics focusing on elucidating the biological function of genes. In this paper，a brief introduction of the content of functional genomics and its mainly new technology ,such as SAGE、ESTs、SSH、microarray、bioinformatics、RNAi and so on were summarized. Meanwhile, it reviewed the developments of functional genomics and simply previewed its prospective applications.
Differential display is the available method used for comprehending the genic expression of different cells or the same cells in different periods or stages. Because of its simplicity, sensitivity, and reproducibility，it has become the most widely used technique for studying differential gene expression This paper summarized the principle of differential display technique and emphasized the relative techniques, which include the extraction and qualification of mRNA, RT, electrophoresis, reamplification，confirmation, sequencing and so on.At last, it introduced the status of this technique used in cancer research,detection of pathology related genes，display of cells growth related genes and the expression of cells gene induced by diferrent factors.
Fluorescent proteins especially green fluorescent protein (GFP) and red fluorescent protein (RFP) are ones of the widest markers in application these years. They can produce fluorescence and were obtained from water biology, GFP was obtained from Aequorea victoria and RFP from Discoma coral. Their structure and luminescence principle are similar, and with the same molecular weight 28kDa. Chromosome marker and plasmid marker are two kinds of way to mark biology. They have been applied widely in protein-protein interactions, screen positive clone, orientation, function and biology of protein factor, ecology of microorganism and so on.
Abstract Somatomedin B domains in different proteins have different functions, including its binding to PAI-1 and to integrins, mediation of homodimerization of protein and inhibition of trypsin activation, et al. SMB domain contains 8 consensus cystines animo acids X4CX3CX9CXCX3CX5CCX5CX5 and the RGD sequence in C-terminal. The disulfide bonds of the recombinant SMB and the SMB isolated by digesting native protein by NMR or protein crystal are not identical.
Mesenchymal stem cells (MSCSs) are progenitor cells of the bone marrow stroma and have capacity of multilineage differentiation. Recently, their functions of immune regulation are being addressed enthusiastically which may relate to various factors: immune molecules expressed on MSCSs surface, their biding bility to immune effect cells and their functions on immune cells. Up to now, acknowledges of the mechanisms of MSCSs immune functions are not consistent. Soluble cytokines, cell surface molecules, IDO, cell contact, cell cycle arrest and antigen presenting cells might involved in the suppression of immune system.
Screening of the cell population after transfection is the key step to obtain genetically-engineering cell lines with the capability for efficient expression of exogenous gene. The development and application of high-throughput sorting methods has significantly improved the efficiency of cell sorting and expanded the range of screening. It resultantly increased the probability of getting the higher produce cell line. In this article, the mechanism and application of high-throughput sorting methods based on fluorescence-activated cell sorting were reviewed, and the prospect of a novel method based on laser-enabled analysis and processing technology was discussed.
The success that therapeutic proteins produced via mammalian-based systems has come with a heavy price burden that is due to inherently high costs of the production of recombinant proteins. The eukaryotic unicellular green alga Chlamydomonas reinhardtii as a new typical bioreactor is getting more attractive in the production of recombinant proteins for several reasons, such as its ability to provide stable chloroplast and nuclear transformants rapidly and its inherently low costs for capitalization and production. This paper reviews the progress of plastid and nuclear transformation in C. reinhardtii. The future prospect of C. reinhardtii as a new typical green factory is also summarized.
The early recognition of the primary tumor and targeting agents to tumor are critical to successful treatment. The primary diagnostic cancer and targeting treatment are still not effective enough, because excellent targeting agents are rather scarce. Peptides possess not only appropriate pharmacokinetic properties to serve tumor imaging or therapeutic targeting agents, but also easily deduce peptide residues using phage display technology. Up till present moment, more than 1000 peptides have been identified targeting to tumors, tumor cells, cognate molecules with metastasis and angiogenic vessels.
The NICE system had been abroad used in Microbiology research ever since Kuipers et al. first brought it forward early in 1995, now this system gained greater promising application and commercial foreground especially when it was used in medical research recently. This mini-review is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over expression of homologous and heterologous genes, (2) metabolic engineering,(3) expression of prokaryotic and eukaryotic membrane proteins, (4)protein secretion and anchoring in the cell envelope, (5) expression of genes with toxic products and analysis of essential genes.
The Study Progress on four Ribosome-Inactivating Proteins have been isolated from the fruit bodies of Flammulina velutipes in recently was reviewed, it contained:four Ribosome-Inactivating Proteins, its properties and functions;action mechanism of Ribosome-Inactivating Proteins ,its applications and prospective.
Protein crystallization is the hot research area in biochemistry and X-ray diffraction is the most reliable method to determine the three-dimensional structure of proteins, but it can only be applied provided suitable crystals are obtained. Obtaining good ordered crystals is currently the major barrier to structure determination. This paper present recent investigation in protein crystallization and emphasize in four major techniques for growing crystals of biological macromolecules: liquid/liquid diffusion ，batch crystallization, vapor diffusion and dialysis, and also mention to detergents and strategy for protein crystallization.
Lactobacillus, a natural treasure, has been used as a kind of probiotics for a long time. Molecular techniques are being employed to modify this Gram-positive bacterium as to better serve food industry, but compare with E.coil, this work is just initial. Recently, Lactobacillus has been developed as a vector for exogenous gene expression to prepare new probiotics. Recent 20 years, Lactobacillus vectors have been constructed and successfully induced to express proteins. As an important component in normal microbial flora in human body, Lactobacillus vectors are very promising. Here, new advances in this aspect are provided.
Bleomycins is an antibiotic compound which is produced by Streptomyces verticillus and extracted from the fermentation liquid. The structure and the biosynthesis of Bleomycins are introduced .The influence of fermentation media , the fermentation conditions and the precursor to the output of Bleomycins is discussed . The methods of the principal components identification of fermentation liquid and the research prospects of Bleomycins are mentioned .
Baculovirus surface display system had been developed greatly since the Yeast surface display was invented. It can be used to display complex eukaryotic proteins requiring post-translational processing including glycosylation and efficient folding for functional activity. The basis of this technology, its development, its applications and perspective are mainly described in this paper.
Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Three kinds of complementary cell lines, HEK-293, PER.C6R, A549 and so on, were described briefly. And the advances in the commercial production of adenovirus vectors were reviewed, including production modes such as batch, sequential batch, fed-batch and perfusion, on-line monitoring and control, purification methods such as CsCl density gradient ultracentrifugation and ion-exchange chromatography.
Abstract:Staphylokinase(SAK) is a 136-amino acid protein,secreted by certain strains of lysogenesis Staphylococci with thrombolytic potential.It combined with Plg to form 1:1 composite, then it made later into Plm and brought into play a role of thrombolysis. As a new thrombolytics, SAK is payed close attention because of high specificity、little bad role of thrombolysis. This review introduced the structure 、biologic activities and their mechanism of SAK, SAK’s gene cloning and expression research concerned.
Diabetes mellitus is one of the heading causes of morbidity and mortality in many countries .It caused by an absolute insulin deficiency due to the destruction of insulin secreting pancreatic cells or by a relative insulin deficiency due to decreased insulin sensitivity, characterized with choronic hyperglycemia. Islet transplantation has been shown to restore normoglycemia ,but a limited supply and graft immune rejection prevents this therapy. Mesenchymal stem cells have the potency of differentiation into islet cells, it may act as a new source of islet transplantation in diabetes mellitus treatment.
The ex vivo expansion of human hematopoietic stem cells (HSCs) is a rapidly developing area with a broad range of biomedical applications. Because the traditional static culture systems can hardly satisfy the ex vivo expansion of HSCs in a large scale, several kinds of bioreactors have been tested to culture these cells, such as perfusion chambers, stirred reactors and rotating reactors. The culture results with these bioreactors are usually much better than the static systems since the bioreactors have lots of advantages.
Transposable elements(TEs) are fragments of DNA that can “jump” in the chromosomes of eukargotes. TEs are classified into two different groups according to their mode of transposition: DNA transposable elements and retrotransposable elements. Recently, two novel families of TEs were discovered by comprehensive analysis of genomic sequences, called MITEs and Helitrons. Customarily, TEs are activated by stress conditions, but many TEs are active to transpose under normal growth conditions. The amplification and the removal of TEs contribute to the genome size variability of plants. TEs can increase the diversity of transposons, create new genes by causing removing, amplifying, unequally cross-over, capturing DNA fragments, or by exon shuffling. TEs scattering within the genome could be the source of the variation required for gene evolution.
This paper is review on the biologic technology of orchid in recent years. It focuses on tissue fostering technology of orchid,seed isolation sprouting technology of orchid,fostering and syncretizing technology of orchid protoplast,and genic engineering of orchid.
The safety of GMF containing cry gene from B. thuringiensis is the heat topic of scientific research and public concern. Here presented the safety research related on starlink, animal food feeded by GMO containing cry, and scientific methods for safety evaluation.
Lactoferrin is an iron-binding glycoprotein with multiple biological functions. In this paper, the structure, mutifunctions and applied prospects of the lactoferrin were reviewed.
Enzyme catalyzed synthesis of polyesters is an eco-friendly and effective technique. By contrast with traditional chemical polymerization, lipase catalyzed reaction has the advantage of high efficiency, high selectivity, mildness. The review highlighted lipase catalyzed polycondensation reactions of simple diacids with diols and activated esters with diols, ring-opening polymerization of lactones leading to the formation of polyesters. The mechanisms of step-growth polymerization and ring-opening were briefly described. Finally, developmental perspective for biodegradable polyesters synthesised by lipase catalyzed was presented.
Phanerochaete chrysosporium can degrade straw (including lignin and cellulose) effectively. It and enzyme produced were used to degrade wasted lignocellulose, which have become the significant and hot point of international academic research. The enzyme system of Phanerochaete chrysosporium was introduced briefly in this paper. The recent research on the optimum fermentation conditions for Phanerochaete chrysosporium and it's degrading straw were mainly reviewed. In addition, some bottleneck problems exited in the recent research and the trend of the further study are presented in the end.
