Objective: To construct a phage display library of single chain variable fragment (scFv) antibodies and to screen a scFv antibody against epidermal growth factor receptor variant type III (EGFRvIII). Method: Balb/c mice were immunized by recombinant EGFRvIIIex and NIH3T3/EGFRvIIIex cell line. Genes of variable heavy (VH) and variable light (VL) chains of antibodies were prepared by RT-PCR from the splenic cells of immunized mice and further artificially joined with a flexible peptide linker, then ligated into the phagemid vector pCANTAB 5E. The phagemides containing scFv were transformed into electrocompetent Hpd3cells. The recombinant phages were enriched through 3 rounds of affinity panning and the anti-EGFRvIII phage scFv clones were identified by ELISA. The fourth round of panning performed at a higher stringency of selection, which used a capture concentration of 0.5μg or 0.05μg. After identified by ELISA, one positive clone was sequenced and transfected into E.coli HB2151 cells to express. Result: A phage display library with a complexity of approximately 7.9×107 was constructed. After the fourth round of panning performed at a increasing stringency of selection, higher affinity clones were obtained. Sequencing analysis showed that the anti-EGFRvIII scFv was 807 base pairs coding 268 amino acids. The soluble scFv exhibited the binding activity to purified recombinant EGFRvIIIex protein and EGFRvIII overexpression cell line. Conclusion: The successful preparation of anti-EGFRvIII scFv will provide an EGFRvIII targeted molecule for the development of antibody-based drugs.
Neisseria meningitidis gene GNA0992 was cloned into the prokaryotic expression vector pET-20b, and soluble expressed in E.coli BL21 (DE3). Purified NhhA stimulated a significant antigen-specific humoral response following three intraperitoneal immunization, while bactericidal assay showed that NhhA can elicit antibodies capable of inducing bactericidal activity against group B N.meningitidis strains. The results indicate that NhhA is a good candidate for vaccine development.
The enhancement of recombinant protein productivity of a stable cell line is essential for developing an efficient large-scale bioprocess. The effect of some media additives and temperature conditions were studied in an attempt to optimize cell growth and protein productivity from a Chinese hamster ovary (CHO) cell line producing human nerve growth factor. The addition of dimethyl sulfoxide (DMSO), sodium butyrate (NaB), KH2PO4 and temperature shift (37℃ and 32℃) were evaluated for the enhancement of hNGF productivity in a fed batch suspension culture. The samples were taken everyday for checking their viable cell density, viability, hNGF productivity and glucose concentration. The culture time was prolonged significantly from 7 days to 14 days when the culture temperature decreased from 37℃ to 32℃. The productivity of hNGF increased in addition of NaB and DMSO though they arrested the cell cycle dramatically. The maximum specific hNGF productivity was achieved in the medium with 2% DMSO cultured at 37℃ and the maximum hNGF productivity was achieved in the medium with 3.65mmol/L KH2PO4 cultured at 32℃. Taken together, the addition of 3.65mmol/L KH2PO4 or 1% DMSO in the culture medium had the greatest effect on hNGF productivity in CHO cells.
Objective: The expression of Ang2 and Tie2 is silenced by RNA interference in order to inhibit the angiogenesis in vitro. The study will supply the theory basis to the further animal research on the inhibition of tumor angiogenesis in vivo and provide the experimental evidence for tumor gene therapy in future. Methods: Using pSilencer 1.0-U6-Ang2/Tie2-siRNA recombinant plasmid to transfect into human umbilical vein endothelial cells (HUVECs), using RT-PCR to analyze the mRNA expression levels of Ang2 and Tie2 in HUVECs of each groups respectively. And the formation of vascular-like structure in HUVECs，transfected by the plasmids ,was studied in vitro by three-dimensional culture. Results: After the recombinant plasmids of pSilencer1.0-U6-Ang2/Tie2-siRNA transfected HUVECs,the mRNA expression levels of Ang2 、Tie2 in HUVECs were average obviously suppressed by RT-PCR（P<0.05）,and there is no significant difference on the expression levels(P＞0.05）between the two plasmids of each siRNA . There is a significant decrease on the quantity and length of vascular-like structure in HUVECs,transfected with the recombinant plasmids by three-dimensional culture model in vitro,（P<0.05）.It proves that angiogenesis has been inhibited significantly in vitro. Conclusions：Ang2-siRNA、Tie2-siRNA can inhibit the mRNA expression of Ang2、Tie2 in HUVECs, and inhibit angiogenesis in the model of three-dimensional culture in vitro.
Enterotoxigenic Escherichia coli(ETEC) is a major cause of diarrhea in animals and infants. The two most important virulence factors of ETEC are colonization factor antigens (CFAs), and the heat stable (ST) enterotoxin or heat labile (LT) enterotoxin.The recombinant plasmid pE3S(S)LK and pE3S(G)LK, containing three STI’s mutants and one colonization factor K99 gene were gotten successfully by PCR and double enzyme cleaving and inserting technology. The result of SDS-PAGE and Western blotting proved that the recombinant strain BL21(DE3)(pE3S(S)LK) and BL21(DE3)(pE3S(G)LK) could produce the fusion protein of 3STI(S)-K99 and 3STI(G)-K99 effectively which were specially recognized by anti-serum of enterotoxigenic Escherichia coli C83922.In addition, the recombinant protein of 3STΙ(S)-K99 and 3STΙ(G)-K99 tuned out to lose their biological toxicity of STΙ(The numerical value of G/C≤0.083) by the test of pouring stomach of sucking mice. It provides basic materials and theoretical introduction for prevent colibacillus diarrhea of newborn piglet and contributes to developing a new effective multivalent gene engneering vaccine.
Angiotensin I-converting enzyme (ACE, EC3.4.15.1) plays an important role in regulating blood pressure. Now, ACE C-domain is identified to be the main site of angiotensin I cleavage in Vivo. In this study, the high expression recombinant Pichia pastoris was constructed and the screening tests was performed in 5 L bio- reactor to obtain the optimal values of several key fermentation parameters. Based on effects on the expression level, the optimal values for the temperature, the con- centrations of methanol and the pH were 26℃, 1.5% (V/V) and 5.5, respectively. Addition of 2%polypeptone to substrate would effectively repress proteolysis. The application of these optimal parameters successfully achieved high-throughput production: the cell density (OD600) of recombinant Pichia pastoris and the yield of crude target protein were respectively 397 mg/L and 446 mg/ L. After the purification with Ni-NTA columns, ACE C-domain was collected with a purity of 98.6%, and the specific activity of it was reached 86 U/mg, which is double fold than that of ACE purchased from Sigma. This provided a zymolytic condition to be used for ACE C-domain in industrial scale production, and provided a high specific activity enzyme for screening specific inhibitor to ACE C-domain.
The C-terminal (comprising 105 amino acids) of neutral amino acid transporter ASCT2 (named as TAIL in this study) is the largest extracellular domain of the protein. ASCT2 is reported to be the main receptor for the envelope of human endogenous retrovirus W family (syncytin) on cell membrane. In this study, ASCT2 cDNA was derived from human breast cancer cell line MCF-7 by RT-PCR method. DNA fragment encoding the TAIL peptide was inserted into pET-41b vector and the recombinant plasmid was expressed in Escherichia coli (E. coli). TAIL peptide, which was recombinated with a glutathione S-transferase (GST) at the N-terminal and a His6 tag at the C-terminal, was expressed in E. coli cells. SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein existed both in the supernatant and the pellet part of E. coli lysates. The supernatant was further purified by affinity chromatography. Highly pure recombinant protein could bind with MCF-7 cells which were reported to express syncytin molecules on their membranes. These results suggest that the recombinant protein has the potential to bind with syncytin molecules.
Interferon alpha are used in clinic to treat a variety of viral diseases and cancers. They have short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of interferon alpha by modifying cysteine residues of the protein with poly(ethylene glycol)(PEG) reagents. But protein seldom have unpaired –SH, so a free cysteine residue was introduced into the protein by recombinant DNA technique and only 1% activity was retained after Pegylation. So a proper position should be selected for Pegylation and enhance the retained activity. Integrated Interferon (IIFN) has a wide variety of biological effects that include viral inhibition, antiproliferation, and immunomodulation . Aspartate residue at positon of 72 was chosed by homology sequence analysisi and space structure simulation methods. Integrated interferon mutant Cys72(IIFN72C) was a cysteine mutant of IIFN. The protein had higher specific activity and may used for site-directed modified by PEG. It was constructed by substitution of Asp at position 72 with Cys using site-directed mutagenesis. The DNA was constructed in pET-23b expression vector, and transformed into E.coli BL21(DE3). IIFN72C was expressed as inclusion bodies with yield of more than 30% of total bacterial protein. The recombinant protein was expressed by auto-induction and purified by DEAE Sepharose FF and Chelating Sepharose FF column chromatography. After purification, the protein was modified with a 20kDa mPEG-maleimide and the mono-PEGylated protein was separated from unmodified IIFN72C by stepwise elution. After purification, the purity of mono-PEG-IIFN72C was up to 98%, and the biological activity was more than 3×107IU/mg, The retained activity was up to 8%, which higher than before. These results confirmed the utility of site-specific PEGylation for creating long-acting interferon with higher activity.
Objective: To construct the recombinant Adenovirus-associated virus(AAV) vector for siRNA targeting HBV mRNA and to evaluate the inhibitive effect on Hep G 2.2.15 cell. Method: The packing cell line(human embryonic kidney 293T cell) was co-transfected with AAV backbone plasmid pAAV-MCS which has been cloned into the selected siRNA , along with the pAAV-RC and pHelper in AAV Helper-Free System. The recombinant Adenovirus-associated virus was packaged and amplified, followed by infection of the Hep G 2.2.15 cells. The expression level of HBV gene and the replication of HBV were analyzed by ELISA and fluorescence quantitative RCR. Result: The recombinant Adenovirus-associated virus vectors containing siRNA targeting HBV mRNA was successfully constructed. ELISA results demonstrated that the siRNA can effectively inhibit the secretion of HBsAg and HBeAg and the fluorescence quantitative RCR results also showed that the copies of HBV DNA and RNA were significantly reduced. Conclusion: The recombinant Adenovirus-associated virus vector can effectively inhibit the expression and replication of HBV in vitro.
The expression of RsPHGPx gene in Pichia pastoris was investigated. The RsPHGPx gene inserted into secretory vector pPIC9K was transformed into Pichia pastoris strain GS115.The singlecopy recombinant strains were screened by G418. In addition, the induction conditions were optimized to get the highest expression of the target protein (the optimum:1%methonal,pH6.0,28℃). Finally,it was shown that the recombinant RsPHGPx could be secreted into the culture supernatant to a level of 102mg/L after 60 hours of induction.The fractional ammonium sulfate precipitation, desalination, and gel chromatography were used to purify protein and more than 90% purity of RsPHGPx was obtained. The bioactivity of RsPHGPx was high-dependent redox-active of GSH and reached its max secreted volume at 60h, the specific activity is 4.2μmol/min·mg. The research has laid the foundation for gaining and exploiting a large amount of RsPHGPx.
The efficiency of photosynthesis can be improved by increasing chlorophyll content in rice leaves. In this study, DET1 （DE-ETIOLATED 1）gene related to high chlorophyll content has been cloned based on the fine mapping and sequence analysis of the DET1 gene was carried out in the high-chlorophyll mutants. The DET1 cDNA full-length of 1536 bp encoded 512 amino acids, and revealed a single T-to-C base transversion in the seventh exon of the DET1 gene which consisted of 11 exons, in comparison with the normal wild-type sequence. This transversion resulted in a conserved Leucine-to-Serine amino acid substitution at the position of 328, and introduced a recognition site for the EcoR I restriction endonuclease and the mutation located in the highly conserved region. Besides, the amino acid sequence of DET1 had more than 62% homology compared to corn and other higher plants. The phylogenetic tree, physical and chemical properties, hydrophilicity and hydrophobicity of the amino acid sequence have been analyzed by the bioinformatics technology. The overexpression vector of pCUPHN-DET1 has been constructed with the binary expression vector pCUPHN. The results laid good foundation for functional verification of DET1.
Objective: To express the Linoleate isomerase gene of Lactobacillus plantarum in Kluyveromyces lactis. Method: Linoleate isomerase （LAI）gene was amplified by PCR from chromosome of Lactobacillus plantarum ZS2058, then the gene was cloned into Kluyveromyces lactis expression vector pKLAC1, the recombinant plasmid pKLAC1-LAI was transformed into Kluyveromyces lactis GG799 by electroporation, The expression level and the activity of recombinant enzyme were detected by SDS-PAGE and Gas Chromatogram . Result: It was demonstrated that a 67 kDa protein which was equal to LAI in molecular weight was secreted in supernatant culture . The analysis of gas chromatography showed that there was a noticeable peak in the retention time of 30.304 min in recombinant broth, the peak was Conjugated Linoleic Acid. Conclusion: It showed that the linoleate isomerase gene from L.plantarum ZS2058 had been expressed and secreted by Kluyveromyces lactis GG799 . Enzyme activity experiments showed that the recombinant enzyme can converse about 26% LA into CLA.
The potential of macroporous resin for separation and purification of lycopene produced by B.trispora were investigated. The adsorptiondesorption properties for lycopene of the resins such as HP20, HP2MG, SP825 and SP207 were studied. The optimal resin for lycopene was screened and selected. The results expressed that HP20 resin had a better adsorption-desorption property for lycopene. It could quickly reach an adsorption balance in one hour. Purification of lycopene using packed bed column by HP20 resin was carried out using step gradient of 55% isopropyl alcohol in acetone followed by 65% isopropyl alcohol in acetone. A 89.55% recovery of lycopene was detected by HPLC. After further recrystallisation the purity of lycopene was above 95% and the structure was confirmed by IR, MS and 1H-NMR. The whole process was simple, easy to operate, with a good industrial application value.
Objectives: To compare the efficiency of HL60 cellular transfection by eukaryotic expressing vector using different methods, and then the most suitable way for transfection could be screened out. Methods: Transfected the empty eukaryotic expressing vector into HL60 cells using 2mm and 4mm electroporating cups respectively using different instrumental parameter, following which the parameter could be ascertained based on the survival rate of HL60 cells. The comparison of transfection efficiency after 48h cellular culture was performed between groups with different plasmid addition and DMSO(dimethyl sulfoxide) supplement. The G418 was employed to screen out positive clones, and its concentration was chosen based on positive rate analysis. At last, pDsRED-C1 empty expressing vector was transfected into HL60 cells following the methods above, and cellular biocharacters could be observed using flow cytometry(FCM), cellular chemical staining and transmission electron microscope. What’s more, the same transfecting procedure was used to transfected another empty eukaryotic expressing vector eYFP into HL60 cells. Results: The death rate of HL60 cells was increased in line with the elevation of intensity of electric shock and frequency of pulse. Square wave was stronger in electroporation than convolution wave. Along with the addition of plasmid, the number of positive transfected HL60 cells was up-regulated at first, and downregulated subsequently with the constant tansfected parameter. Moreover, the positive transfected HL60 cells was much more in 2mm electroporated cup than 4mm electroporated cup under the same plasmid addition. The supplyment of DMSO in refrigerated transfection system could significantly improve the positive rate of transfection about nearly 13 times. 400μg/ml G418 was choosen as the best screening drug concentration. After cellular electroporation, the cellular surface antigen CD11b and CD14 could not be detected, and that, the apoptosis could not be found by observation of cellular morphology and ultramicrostructure. Same procedure was successfully used in the transfection of eYFP-C1 into HL60 cells. Conclusion: The modified electroporating procedure could significantly promote the positive rate of cellular transfection, which would lay the foundation of researchers’ following experiments.
A simple, rapid, and low-cost method to identify transgenic tobacco plants carrying a single copy of integrated DNA by one-step genomic polymerase chain reaction was described. The reaction employs one set of primer pairs that amplify the target gene (NPTⅡ) derived from the integrated T-DNA together with the know endogenous single-copy gene (RNR2) as a reference under the same melting temperature in a single PCR. After PCR amplification of the genomic DNA from transgenic tobacco plants, two expected bands were observed. When the band intensity ratio is one, this means that the transformants are carrying a single copy of the integrated gene. It is further observed that the expected 3∶1 Mendelian ratio was obtained in all single-copy T-DNA transgenic lines.
Phage display technology was used to select transferrin binding peptides (TfB) from a phage fused random heptapeptide library. After three rounds of biopanning, positive recombinant phage clones were enriched. For estimation the efficiency of phage display screening the binding peptides, the selection criteria of recovery yield and specificity ratio in the panning process were developed. The affinity constants of the four most positive TfB clones were assayed with the method of the measurement of phage titres, and the corresponding TfB sequences were obtained through DNA sequencing. Because of transferrin receptor as a tumor biomarker, transferrin has been used as a drug carrier to deliver therapeutics targeting cancer cells. Therefore TfBs will be explored as useful protein tags for binding Tf to improve the transport properties of recombinant protein drugs in vivo.
IFN-λs(including IFN-λ1, IFN-λ2, and IFN-λ3)is a newly identified IFN family whose characters related to the type I IFNs and IL-10 family members. IFNλs act through a cell surface receptor composed of two chains, the first one (CRF2-12/IFN-?R1/IL-28Rα) being IFN-λspecific and the second one (CRF2-4 / IL-10R2) shared with IL-10. IFN-λs signal through the IFN-λR1 and activate JAK-STATs pathways, as well as type I IFNs. So, IFN-λs exhibit several common features with type I IFNs: antiviral activity, antiproliferative activity and in vivo antitumour activity.And most importantly，clinical trialsⅠwith PEG-IFN-λ1was completed as a novel medcine to HCV. In this review, we summarize the lasted research progress about the biology of IFN-λs and point that IFN-λs may be applied to the clinical medicine in the near future.
PEGylation, the covalent attachment of polyethylene glycol (PEG) to proteins, has been used as an effective strategy to overcome several shortcomings of proteins for therapeutic uses. The therapeutic efficacy of PEGylated protein drugs can be improved through prolonging their circulating half-life, reducing their immunogenicity and proteolysis, and increasing their stability and solubility. Furthermore, site-specific PEGylation is an attractive approach for the maximizing the bioavailability of drugs, because a homogeneously modified product with high activity retention and yield can be achieved. Recent advances in site-specific PEGylation were reviewed. The site-specific PEGylation strategies and suitable modified sites were particularly introduced. Finally, the future trends and prospects in site-specific PEGylation were discussed.
Biocatalysis was employed to do chemical transformations on non-natural man-made organic compounds. Biological methods offer a true practical advantage over chemical synthesis. With the development of microbial strain involved in bioconversion, enzymes and proteins are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. Recombinant microbial whole-cell biocatalysis is a valuable optimization and modification approach for producing enantiomerically pure interemediates. Metabolic engineering based on the systems-level analysis of cells and organisms is now offering a new powerful way of designing and developing strains to improve the performance in biocatalysis. Recent advances and development strategies of bioconversion were highlighted here.
Aspergillus oryzae expression system has many advantages when compared to E.coli and yeast expression system. At present the vast majority of studies focus on how to improve the production of protein by A. oryzae in liquid submerged fermentation.However, in solid culture conditions, the ability of A. oryzae to produce a variety of proteins was stronger than in the liquid culture conditions. This is not only related to growth patterns of A. oryzae under different culture conditions, but also with the characteristics of regulation factors, such as promoters, which control the specific gene in different metabolic pathways. The expression efficiency of A. oryzae glucoamylase gene, glaB, under solid culture conditions was significantly higher than that of the liquid culture conditions. The glucoamylase gene was used as an example and the studies of glaB promoter were overviewed, which provide a reference for making better use of such promoters in the future.
Transcription Mediated Amplification is a target nucleic acid amplification method that uses RNA polymerase and reverse transcriptase to produce RNA amplicon from a target RNA or DNA under isothermal condition at about 42℃. TMA produces 100~1000 copies per cycle which results in a 10 billion fold increase of copies within about 15~30 minutes. TMA is now widely used in genotyping of human leukocyte antigen and diagnosis of many bacterium and viruses.
Alkaline pectinases are a group of pectinolytic enzymes displaying optimal activities at alkaline pHs, which characteristic makes it feasible for them to be promising biocatalysts used in the textile and paper industry. The molecular characteristics and catalytic properties of native and recombinant alkaline pectinases from bacteria were summarized and discussed. The latest applications on bioscouring, biopulp making and induction of plant disease resistance were also stated.
NPGI was established in 1998 as a coordinated national plant genome project. The NPGI is governed by the Interagency Working Group on Plant Genomes (IWG-PG) under the auspices of the National Science and Technology Council Committee on Science. NPGI participating agencies: US Department of Agriculture (USDA). Department of Energy (DOE), National Institutes of Health (NIH), National Science Foundation (NSF), Office of Science and Technology Policy (OSTP), Office of Management and Budget (OMB) and the United States Agency for International Development (USAID). IWG developed a five-year strategic plan for the National Plant Genome to guide and coordinate genome research.The situation of Plant Genome Research Program supported by National Science Foundation in 1998~2009 was analyzed.It includes the objectives, funding, projects, etc. Finally got some inspiration.
