Objectives: To clone immunoglobulin J chain (IgJ) gene, polymeric immunoglobulin receptor (pIgR) gene and human immunoglobulin A heavy chain constant region coding sequence (IGHA) for construction of secretory immunoglobulin A expressing plasmids. Methods: According to the "Genomic DNA Splicing" technique established in our lab, highly efficient exon primers were designed by software to amplify the exons directly from genomic DNA extract. An overlapping PCR was then performed with manually designed overlapping primers to join adjacent exons together to form a full-length coding sequence. The full-length PCR products were purified and ligated with pGEM-T Easy Vector. After transformation, clones were screened and positive cloned were subjected to sequencing. Results: The full-length PCR products had the expected molecular weight and sequence analysis confirmed that the cloned sequences were identical to the relative entries of the GenBank database. Conclusion: IgJ, pIgR and IGHA genes are successfully assembled by the "Genomic DNA Splicing" technique, which suggests that this technique could be a reliable strategy for cloning of multiple-exon cDNAs.
Objective This experiment research on the influence of protein expressed by hTERT gene on the NSCs biological characteristics. Method In this experiment, constructed recombinant plasmid pEGFP-N1-hTERT was transfected into rat fetal NSCs with lipid-mediated transfection method Transfected NSCs differentiation in vitro tests, tumorigenicity in nude mice test, RT-PCR and Western-blot analysis and flow cytometry. Result Transfected NSCs stem cells still have the potential growth characteristics and multipotency in vitro, no tumorigenic, and transcription product of hTERT gene mRNA and hTERT-GFP fusion protein expression in the transfected NSCs. Conclusions The transferred NSCs of telomerase activity was up-regulation and the proliferation of cells were promoted in vitro by hTERT gene, and also displayed immortalization tendency.
AIM：in oder to clone endothelin 1(ET1) gene, express ET1 fusion protein , induce organism producted ET1 antibody, and neutralize superfluous ET1 in patients. METHODS：The gene of human ET1 was synthesized according to the preferential codons of E. coli, cloned into the EcoRI and SalI sites of vector pThioHisA. The recombinant plasmid pThioHisA-ET1 was constructed , sequenced and transformed into E.coli TOP10. Induced and expressed fusion protein were identified and analysed by 12% SDS-PAGE and densitometry analyses. After the elution, denature and renature, the fusion protein Thioredoxin-ET1 was obtained by ProBondTM chromatogragraphy. The purity of Thioredoxin-ET1 was detected by HPLC. Inoculate Thioredoxin-ET1 once per mouse every 2 weeks in 25, 50 and 100μg separately on 3 groups for 4 times. 10 days after last inoculation, we obtained venipuncture blood. RESULTS：SDS-PAGE and densitometry analyses showed the expressed fusion protein Thioredoxin-ET1,with a molecular weight of about 19 kD, was about 50% of total bacterial protein after induction at 37℃ by 1mmol/LIPTG for 4 hours. The purity of purefied Thioredoxin-ET1 is 90%. The results of Western blot and ELISA indicated that Thioredoxin-ET1 fusion protein has immunogenicity of ET1 and the titer of ET1 was about 104 in anti-serum. CONCLUSION： Thioredoxin-ET1 fusion protein could induce ET1 antibody production in mouse. And it could be used to explore mechanisms of ET1 over-expression in patients and provided a new strategy to immunotherapy.
herpes simplex virusⅠ(HSVⅠ) regulating the pathway of transcription and translation modify in host cell is a very systematic and complicate system. A clear understanding of the concrete mechanisms of infection will greatly help to comprehend the virus replication and the interaction with the host cell. By the analysis of 2-DE, the heterogeneous nuclear ribonucleoprotein H2 in human fetal liver cell represent distinction after the HSVⅠinfection.Utilization of Northern blot and Western blot technologies verified the expression of hnRNP H2 in different stage of virus infection is varied.
Objective To screen the interactive proteins of hCLP46 and provide clues for the studies on hCLP46 function and pathogenesis of MDS-AML. Methods The human leukocyte cDNA library was screened with with pGBKT7-hCLP46 as bait plasmid by yeast two hybrid system , and Leu+, Mel+, Trp+, His+ positive yeast clones were obtained , The inserted fragments of identified positive clones were sequenced and analyzed with Blast in NCBI. Results There are 86 positive yeast clones which are obtained through screening the human leukocyte cDNA library with pGBKT7-hCLP46 as bait plasmid. Four different candidate gene sequences are obtained by Blast analysis in NCBI. Conclusion Four gene sequences were obtained , these coding proteins can interact with hCLP46 ,which may be involved with pathogenesis of MDS-AML.
Tapping panel dryness (TPD) occurrence in high latex yielding rubber tree (Hevea brasiliensis) is characterized by the partial or complete cessation of latex flow upon tapping leading to severe loss in natural rubber production around the world. To better explore and understand the mechanism of Tapping Panel Dryness(TPD) of Hevea brasiliensis onset, differential proteomic analysis is conducted in C-serum proteins from latex of healthy and TPD trees by two-dimension gel electrophoresis (2-DE) .The C-serum proteins from latex of healthy and TPD trees were separated by immobilized pH gradient(IPG) based 2-DE. The commassie brilliant blue-stained 2-DE were scanned with digital Image Scanner and analyzed with PDQuest 7.40 analysis software. After in-gel protein digestion , peptide mass fingerprint ( PMF) of different-expressed protein spots were obtained with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching were used to identify proteins in Swiss-Prot and NCBInr database by Mascot software. Results: ①Average protein spots were 1075±35, 1134±27 in C-serum proteins from latex of healthy and TPD respectively, the matched spots were 982±38,1008±22 in both the two types of tissues respectively , and the average matching rate was 91.89﹪,88.72﹪ respectively. ②A total of 970±25 protein spots was matched between the 2-DE map of C-serum proteins from latex of healthy and TPD. Forty spots with changed expression level remarkably were measured by MALDI- TOF-MS and 27 proteins were identified. In this study, the well-resolved and reproducible 2-DE patterns of C-serum proteome from latex of healthy and TPD trees were established and some different-expressed protein spots between the two types of tissues were detected by mass spectrometry. These proteins may be involved the onset of TPD syndrome. The results presented here demonstrate that 2-DE technique provides a powerful complementary approach for the identification of TPD related proteins from rubber tree.
Objective: To separate and purify the Angiotensin Converting Enzyme Inhibitor(ACEI) named hypotensive factor(HF) from Naja naja atra venom and determine its biological activity. Methods: HF of snake venom extract was isolated and purified by Sephacryl S-100 gel filtration, and CM Sepharose F.F. ionexchange chromatography respectively. The purified product was identified by HPLC. The molecular weight was determined by SDS-polyacrylamid gel electrophoresis. The ACE inhibitor activity was determined by UV spectrophotometer. The effect of HF on contraction by BK was tested by using isolated intestinal smooth muscle from rabbit. Results: SDS-PAGE showed a single band of HF and a molecular weight of 8.2kD. HF was a single polypeptide chain and consisted of 12 amino acids. Recovery rate of protein was 5.70%. ACE was inhibited by HF obviously. The inhibitory rate has a positive correlation with the concentration of HF．The IC50 of was1.02?g/ml. HF could strengthen the contraction of isolated intestinal smooth muscle from rabbit by BK. Conclusion Our method has been proved to be successful for the purification of HF from Naja naja atra venom. HF has a similar effect as Angiotensin Converting Enzyme Inhibitor, and shows a significant inhibitory effect on ACE.
Objective To develop a high efficient expression, purification system of recombinant Arginine deiminase(ADI).Methods cDNA fragment encoding for mycoplasma ADI was obtained by artificial synthesis and was cloned into prokaryotic expression vector(pBV220). The recombinant ADI was generated by the transformation of the recombinant vector into the host strain DH5α. Anion exchange and gel filtration chromatography was carried out for purification of the recombinant ADI. The biological activity of final product was detected by the assay of agrinine degradation in vitro. Results A prokaryotic expression plasmid pBV220-ADI was generated successfully, and was identified by DNA sequencing; The recombinant protein was highly expressed in DH5α, the proportion of the recombinant protein is exceeded 35% of the whole protein. The inclusion bodies were solubilized with 6 M guanidine hydrochloride under reducing conditions in order to avoid incorrect disulfide-bond formation of the recombinant ADI molecules. Dilution and dialysis at lower degrees temperature were the optimum renaturation methods. After gel filtration, the purity and specific activity of rADI reached 95% and 80 IU/mg respectively. Conclusions A set of protocols for high efficient rADI expression and purification has been established, which is simple, efficient and applicable.
The differences of photosynthetic characters and yields among three leaf-types of Pinallia ternate, the willow-leaf type, the peach-leaf type and the heart-leaf style, were studied and compared. The result showed that P. ternata was a typical shade-plant, of which LCP was only 700μmol•photons/s•m-2. Photo-inhibition was observed among all the three leaf-types of P. ternata, and the willow leaf type showed the most obvious photo-inhibition. Both of the light response Pn curve and single tuber weight were significant different among leaf styles. Result for studying the chlorophyll fluorescence parameters showed that all the difference of NPQ, qP and qN among three leaf-types of P. ternata were significant and the difference of potential-photosynthetic productivity among them was significant. The willow leaf style had the strongest light-tolerance capacity. We found that individuals of T2(peach-leaf type) had the highest Pn and single tuber weight among 11 superior individuals and the individuals of L2(willow-leaf type) showed the strongest light-tolerance capacity.
The human canstatin cDNA was amplified by RT-PCR and then directionally cloned into pUΩ expression vector. The recombinant pUΩ-Can vector was connected with the screening marker (bar box), to construct a eukaryotic expression vector called pUΩ-Can-Bar. This expression vector was introduced into the D. salina by glass beads method. The screening culture of transformants of D. salina was performed in solid media containing 5 ?g/ml PPT, and the analyses of transformants were carried out through PCR and Southern blot. PCR results revealed that specific 700 bp products were detected in the different transformants of D. salina but not in negative control. Southern blot analysis further demonstrated that human canstatin gene was integrated into the D. salina genome. Moreover, the results of genetic stability analyses of transformants demonstrated that canstatin gene was stably inherited in the D. salina transformants. The successful preparation of the D. salina transformants will provide the experimentation evidence for producing canstatin protein cosmically by using the D. salina bioreactor and give a better prophase work basis for clinic application of canstatin protein early．
With the development of functional genomics, high throughput analysis of genes' function has been the mainstream of research, and exogenous gene's over expression via Agrobacterium-mediated transformation is the most commonly used method in gene functional analysis. In this study, the versatile plant expression vector cassette named pBHT-5 was constructed by the method of site-specific mutagenesis based on pBI121. First of all, the restriction enzyme SfiI recognition site in trfA gene (X00713) which was relevant to plasmid replication and stability was replaced without changing its amino acid composition. And then the SfiIA、SfiIB sites were added between promoter CaMV35s and terminator NOS. The versatile plant expression vector cassette we got can be directly used to construct plant expression vector containing the full-length genes cloned by Clontech SMARTTM technology, which will raise the efficiency of vector construction. The result will provide basis of new genes' high throughput screening and functional analysis, then get the new genes functioning in plant osmotic stress resistance.
A 501bp sequence of Sus scrofa IFN-αgene and Susscrota domestica of changbai IFN-αgene was amplified by normal polymerase chain reaction (PCR) from their genome whose homology are more than 98.4% ，compared with nucleotide sequence published on NCBI .Referring to Sus scrofa IFN-αgene sequence,a new sequence was synthesized after codon bias and mRNA structure optimizaed .Then ,this sequence was inserted in pWL vector and the recombinant plasmids were transformed into the E.Coli DH-5α.After fermentation,the expression level of recombinant protein are 25% by SDS-PAGE analysis.The biologic activity was 4.45×106 IU/mg by VSV-WISH system analysis after protein refolding and purification.
Nano materials are popular with its interesting properties. Large specific surface area, high mechanical strength, good absorbability make them good potencial carriers of catalyst. With the help of chemical modification, we used nano SiO2 particals as carrier to immobilize β-glucosidase with covalent binding. It is found that the load of nano particals can reach 1/8 of its weight, and the recovered activity of enzyme is 70%. The resistence of immobilized enzyme to organic solvent wase improved obviously, which was then used in Ethyl Acetate-Water-two-phase-system to hydrolyzed soybean isoflavones.
To develop an oral vaccine against Helicobacter pylori infection, the H.pylori napA gene, encoding the neutrophil-activating protein (NAP), was expressed in a live delivery vehicle Lactococcus lactis. And the immunogenicity of the recombinant NAP was evaluated after the lactococcus lactis was administrated orally into mice. First, the napA gene of Helicobacter pylori was cloned into the prokaryotic expressive vector pNICE:sec. Second, the recombinant vector pNICE:sec-nap was transformated into Lactococcus lactis strain NZ9000 to express NAP protein. Then the recombinant NAP was induced to express and was identified by SDS-PAGE and Western-bloting. Finally, ICR mice were randomly divided into 4 groups and administrated orally with PBS,L.lactis pNICE:sec, L.lactis pNICE:sec-nap, and the inactivated H.pylori respectively. The specific IgG and IgA were identified after 7 times immunization. The results in this experiment were described as follows. The napA was amplified and cloned in the vector pNICE:sec successfully. The fusion protein (17kDa) was expressed in L.lactis by the induction of the nisin. The quantity of expression accounted for 9.5% of the total bacterial protein. Western blot analysis confirmed that fusion protein could be recognized specifically by the serum of anti-H.pylori. AntiNAP IgG titers in the serum of L.lactis pNICE:sec-nap group was higher than the L.lactis pNICE:sec group and no obviously difference with the inactivated H.pylori. The anti-NAP IgA titer in the intestinal mucosa of L.lactis pNICE:sec-nap group was higher than other groups. These results suggest that the recombinant L.lactis which expresses napA, may be applicable as an oral vaccine to induce protective immunity against H.pylori.
TThe active sites of β-fructosyl-transferase from Aspergillus oryzae GX0011 were investigated by means of chemical modification and kinetic study. NBS(N-bromosuccinimide)、PMSF(Phenylmethyl-sulphonyl fluoride)、EDC(Carbodimide) seriously inactivated the enzyme but not in the presence of substrate. The inactivation reactions followed pseudo-first-order kinetics. Further kinetic analysis of the inactivation indicated that at least one serine/threonine residue, one tryptophan residue and one asparic acid/glutamic acid residue might be involved in the active site of the enzyme. PCMB(p-chloromercuribenzoate) and TNBS(2,4,6-trinitrobenzene sulfonate) could inactivate the enzyme even in the presence of substrate. This implied that cysteine and lysine residues might not be in the active site of the enzyme but involved in maintaining the conformation of the enzyme. DEPC(Diethylpyrocarbonate)、AA(acetylacetone) and NAI (N-acetyl-imidazole), did not obviously influence activity, thus eliminating the possibility that histidine, arginine and tyrosine participated in catalysis.
Klebsiella oxytoca ME-UD-3-4 with high glucose tolerance was obtained from the original strain ME-UD-3 after mutagenesis by UV coupled with diethyl sulfate and following continous subculture in the broth with gradually increased glucose concentration. Compared with the original strain which could only endure 120 g/L glucose, the mutant was able to tolerate as high as 300g/L glucose. 95 g/L glucose was depleted by the mutant in 40h,which was 8h shorter than that of the original strain; and the final 2,3-butanediol concentration and productivity of which achieved to 43.0g/L and 1.08 g/L·h, respectively, with the yield up to 91% of the theoretical yield.
Staphylococcus aureus is a primary pathogen that poses a serious health threat as a consequence of multiantibiotic resistance. S. aureus can cause diverse serious diseases in humans and animals through the production of toxins, most toxin genes are positively controlled by the accessory gene regulator (agr) system which is responsible for the regulation of more than 30 virulence factors in S. aureus. The agr system which could be a useful target for drug selection is one of the most extensively studied quorum sensing systems for its potential relevance to human disease. Yet many questions regarding the agr signaling remain unsolved. Herein, we review the agr system as we currently understand them and discuss the inconsistencies of the role and fundamental mechanisms of agr in staphylococcal infections.
Cationic antimicrobial peptides are a kind of peptides with anti-extrogenous pathogen activities. They are derived from biological defence system. Most peptides share common features such as small size, strong heat steadiness, wide antibacterial spectrum and special antibacterial mechanism, which are different from other general antibiotic. According to the research report, this paper overviewed sorts, antimicrobial mechanism, design and synthesis of cationic antimicrobial peptides.
HBeAg is an ungranular secretory protein, which encoded by C gene of HBV DNA and it increases with the replication of HBV. So it is one of the markers of active replication of HBV in clinical diagnosis. HBeAg is important biologic raw materials which is widely used in preparation of related diagnostic articles with HBV infection serological detection. The technology of expression and purification of recombinant HBeAg is quite mature, which had successfully expressed the target protein in various expression systems. The key factors on HBeAg expression include important site mutation in precore region ,the choice of vectors, effects of RNA interference（RNAi）and so on. Therefore, in order to meet requirements of related diagnostic products, it need to improve expression level and purity of recombinant HBeAg and avoid cross-reaction with HBcAg. In a word, it showed that acquisition of high quality recombinant HBeAg could lay substantial foundation for improving diagnostic products, provide a reliable evidence for exploiting new type of theapical and preventive HBV vaccine and offer possibility of HBeAb detection methodological optimization.
For the important role in economic production, the study has been focused on the technique of cryopreservation of boar semen. During earlier time the study mainly focused on screening and optimizing the cryprotective mediums and freezing methods, which exploited the technique of cryopreservation deeply. However, the study indicated that the fecundation ability was decreased no matter what cryoprotective mediums and what freezing methods were used, companied with the losing of proteins, changing of proteins expressing quantity and increasing or decreasing of the function and activity in sperm after cryopreservation. Now, the study is altering to exploit the essence of the change of protein structure and function, and elucidate the cryodamage mechanism of sperm cryopreservation, which may provide the basic theory for cryopreservation of boar semen and promote the rapid development of the technique of boar semen cryopreservation.
Aphids have been one of the most serious threatens to China's agriculture, being responsible for the direct damage to crop and the transmission of plant viruses under the present environment. With the in-depth understanding of life science and the development of transgenic technology, genetic engineering in developing aphid-resistant plants has already had attention and been proven to be an effective way to resolve the problems caused by aphid pests. The progress is depending on the characterization of genes which have high toxicity or inhibition activity to aphids and the culture of aphid-resistant transgenic plants. This review describes the research on aphid-resistant genes used in current study and application, and will focus on the expression of Galanthus nivalis Agglutinin gene and Amaranthus caudatus agglutinin gene in transgenic plants and their aphid-resistant effects.
Listeria monocytogenes (L. monocytogenes) is an important human foodborne pathogen responsible for listeriosis. It is one of the hot topics in food safety area on how to detect L. monocytogenes rapidly and effectively. In recent years, its detection assays have a rapid development. The purpose of this review is to summarize the culture-dependent enrichment, immunoassay-based and nucleic acid-based methods for detection of L. monocytogenes at present. Lastly, the review introduced the new strategy of detection assays.
Switching Mechanism At 5' end of the RNA Transcript (SMART) is a technology used in biology researching, So far, there is no review only about SMART technology. So, this review tries to investigate the research developments of principles, methods and applications of the SMART technology. Based on some researches and combined with the review of the related literature at home and abroad, the authors analyzed and evaluated the latest development of the research on SMART. As the applications of the SMART technology expand in many fields day after day, it has been proved that the SMART technology is a very useful and efficient skill to construct full length cDNA library. As more and more researchers know this technology, the advantages of the SMART technology become obvious, and meanwhile the disadvantages of the SMART technology also show up. That is to say, the technology needed to be improved.
2,3-butanediol(2,3-BD) is an important chemical intermediate and can be used in many fields. 2,3-BD production come to a peak during World War Ⅱ because 1,3-butanediene used for synthetic rubber production could be made from 2,3-BD. Due to the increased demand for polybutylene terephthalate resin, gamma-butyrolactone, spandex and their precursors, the demand and yield of 2,3-BD increased stably in recent years. Although 2,3-BD production by fermentation has been studied widely for many years, its industrialization did not realized till now. The microbe species and the physiological function of 2,3-BD to cell，metabolic pathway, mechanism of stereoisomers formation, the factors influencing the fermentation and downstream recovery of 2,3-BD is discussed, and some problems should be solved in 2,3-BD production is also presented in this article.
