With the rapidly development of the biotechnology industry, large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications. Bacterial cells are most often used for the production of recombinant proteins. However, recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive. To circumvent this problem, several eukaryotic expression systems have also been developed over the years, ranging from yeast to mammalian cell-based technologies. For many mammalian proteins, especially those secreted and modified posttranslationally, a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells, i.e., mammalian cells. Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins. Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest. As it is allowed for easy and efficient purification, many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted. The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells. The noncatalytic C-terminal hemopexin-like domain of MMP-2, PEX, can block angiogenesis and tumor growth in vivo. Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties, as well as for their industrial applications. For this purpose, the rat growth hormone, mouse IgGκ chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells, and their secretion efficiencies were compared by Western blotting and ELISA. Western-blotting of PEX protein from culture media, resulted in detection of proteins with the predicted molecular mass, which indicate that all of the signal sequences could direct PEX secretion successfully. The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency. Thus, expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly. To examine whether the observed difference in secretion yields is promoted at the transcriptional level, a RT-PCR analysis was performed at 6 h after transfection. The presence of mRNA transcripts of PEX was observed in all the DNA constructs. Moreover, semiquantitative reverse transcription (RT-PCR) results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection. Though there was no difference in the expression levels of PEX at an early time point after transfection, the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells. Hence, the described difference in exported yields is probably promoted at the secretion level, rather than at the transcriptional level. Chick chorioallantoic membrane (CAM) bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis. The MMP-9’s signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.
It is well documented that altered biosynthesis of cell surface N-linked oligosaccharides is associated with the transformed cells and tumors. N-acetylglucosaminyltransferase II (GnT-II; EC 2.4.1.143) is a medial Golgi enzyme that catalyses the incorporation of a GlcNAc residue in β-1,2 linkage to the Man-α-1,6 arm of the N-glycan core. This is an essential step in the biosynthetic pathway leading from hybrid to complex N-glycans. Because functional GnT-II is an prerequisite of N-Acetylglucosaminyltransferase V performance, we speculated that GnT-II was involved in cancer development and progression. In this study, the expression of GnT-II in mouse breast cancer cells 67NR and 4T1 which have different behavior of metastasis was analysed using RT-PCR. The amounts of GnT-II in the highly metastatic cell 4T1 increased to 1.53 times of the lowly metastatic cell 67NR. To determine the association of GnT-II with tumor progression, the GnT-II encoding gene was amplified with RT-PCR and cloned into retrovirus vector pMSCV, resulting in pMSCV-GnT-II. The recombinant plasmid was transfected into 4T1 and the transfected cells were selected in the medium containing puromycin, which were harvested to detect the adhesion ability to fibronection and the migration potential by transwell system. The cell adhesion to fibronectin was weakened by 67% and migration potential was increased by 82%. Our data indicates that GnT-II mediates cell adhesion and migration, thus may play an important role in cancer metastasis.
Objective: To investigate the effect of high-concentration RA (all-trans retinoic acid，RA) on the differentiation of ESC. Methods: During the formation of embryoid bodys, exposured to 5μM RA. Neural precursor cells were replated in different substrates. Immunofluorescence analysed the different stage of ESC differentiation. Results: 5μM RA treatment of differentiating ESC was important for the induction of nestin positive cells. Approximately 76% of the cells were β-tubulin III positive, 55% of β-tubulin III positve cells were GABA positive when cultured on LPO, whereas only 4% were CHAT positive. Conclusion: High-concentration RA strongly drived neural induction.
Resistin is a newly discovered adipocyte hormone.With the method of gene engineering,we got resistin.The RSTN gene was subcloned to the expression vector pET-32a(+) and the recombined plasmid named pET-RSTN was constructed successfully. The plasmid pET-RSTN were transformed into the Eoli.BL-21(DE3). After inducing by IPTG the fusion protein of resistin were expressed and its molecular weight was about 30kDa. As temperature、IPTG concentration and induction time were optimized,the soluble recombinant protein was high expressed at 30℃ for 4h with 1mmol/L IPTG induction. The product was identified by SDS-PAGE and western-blotting. The expression production purified on Ni2+-NTA column.
In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2 (Gln49-PLA2), site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR. Aspartic acid 49 phospholipase A2 (Asp49-PLA2--Q49D-PLA2), the mutant of Gln49-PLA2 was expressed in E. coli with pET32a+ vector. The fusion protein, expressed as inclusion body, after being denatured, was on-column refolded and purified by immobilized metal affinity chromatography (IMAC), and then cleaved by Factor Xa. The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography, with the recovery rate of 1.3%, and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg. It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding, especially in rich disulfide bonds conditions.
Abstract Alfimeprase(ALF) is a recombinantly modified variant of non-hemorrhagic zinc metalloproteinase fibrolase. By digestion with the clone vector p43-alf, the target gene alf was obtained and cloned into the Pichia pastoris expression vector pPICZα A, through high efficiency transformation and Zeocin selection, the recombinant strains of pPICZ?A-alf / GS115 were obtained. After culture parameters of pH value, methanol daily addition concentration, cell density and methanol induction time points were optimized, the production of recombinant Alfimeprase (rALF) reached up to 425 mg/L. By His•Bind chromatography purification, the purity of secreted rALF was as high as 95 %. SDS-PAGE and western blot analysis showed that rALF had a molecular weight of 24 kDa and bound specifically to mouse anti-His• tag monoclonal antibody, activity identification results of the modified fibrin plate method also demonstrated that the secreted rALF had high fibrinolytic activity. Thus the report set up an important foundation for further studies and industrial production of ALF.
The B.subtilis ywtD gene, encoding a γ-polyglutamic acid (γ-PGA) depolymerase, was amplified from the genome of B.subtilis NX-2 by PCR.The comparability between the cloned ywtD gene sequence to the reported sequence is high to 99.0%. Only one of the substituted nucleotide base caused the change to the amino acid sequence. The recombinant plasmid pET-15b-ywtD was then transformed into E. coli Rosetta (DE3) and the ywtD gene product could be expressed with the induction of 0.5mmol/L IPTG. The YwtD protein exhibited a remarkable activity in γ-polyglutamic acid degradation. The molecular weight of γ-PGA could be reduced from 700kDa to 20kDa after 72h through the enzymatic hydrolysis and consequently trended to be constant.
The selective side-chain cleavage of phytosterol to 4-androstene-3, 17-dione (4-AD) and 1, 4-androstadiene-3, 17-dione (ADD) by Mycobacterium sp. was described. Because of the similarity in chemical structure between 4-AD and ADD, it is difficult to separate them from the fermentation broth. So far, it has been verified that the ADD can be produced by dehydrogenation of 4-AD.In this reaction, 3-Ketosteriod-1-Dehydrogenase (ksdD) plays an important role. In this research, the gene knocking out method was used to solve the problem. In this research, partial sequence of ksdD was obtained by PCR which was 631bp in length. Then ,a targeting vector pUC19-MK was constructed, which was electroporate into the original strain Mycobacterium neoauru. The method of homologous recombination was used to knock out ksdD gene located in the chromosome of Mycobacterium neoauru. In this way, ksdD would lose its enzyme activity. In the result, 5 transformants were screened. The experiments of steroid transformation by the transformants were carried out. The productivity of 4-AD reached 17.52％ after 144h, which is 192% higher than the original strain. Meanwhile, the productivity of ADD reached 6.12%, which is 89.9% lower than the original strain.
The Telomeric Repeat Amplification Protocol (TRAP) and its modified versions change the size and/or the ratio of the telomerase products in the amplification stage of the assay. Based on TRAP, we developed a useful method for detecting telomerase activity in rice. In this report we designed a special precursor primer and a special reverse primer and conducted two steps of PCR cycles. GENE Genius? Bio-imaging System was applied for this quantitative analysis for exploring telomerase activity and its optimal reaction conditions. The method ensured that the optimal reaction conditions for the telomerase was 19℃,13minutes, at a concentration of 0.28 u g/μ l. A quantitative analysis method was established for detecting telomerase activity in rice. With this method, we detected telomerase activity in roots, young leaves and young panicles of six parental lines of hybrid rice. The results show that young panicles have the highest telomerase activity, demonstrating that telomerase activity is closely related to the cell vitality in plants.
The effects of six vitamins on a novel isolate, Corynebacterium glutamicum SYPS-062, which could directly produce L-serine from carbohydrates were investigated. These vitamins play the important roles in the L-serine metabolic pathway. By comparing the L-serine production, biomass accumulation, transformation efficiency (YP/S) and L-serine yield per biomass (YP/X), the L-serine metabolic regulation mechanism by each vitamin was analyzed. and the results show that all these tested vitamins－VB1, biotin，VB2，VB6，folate and VB12 could enhance the growth of C. glutamicum SYPS-062 and improve the production of L-serine, however, the mechanisms of their metabolic regulations were different. The obvious decrease of YP/S and YP/X reflected that the addition of VB1 and biotin caused more L-serine to degrade to pyruvate by accelerating the transformation of pyruvate to TCA circle, and the promotion of VB6 on L-serine degradation was more remarkable than the synthesis, but the significant increase of biomass led to the increment of the total L-serine production by adding above three vitamins, which was 31%，28% and 11% higher than the control; VB2 could improve both the biomass (35%) and the L-serine production (33%) equally, due to the promotion of the energy metabolism, and thus it does not influence the flux distribution of metabolic pathway of L-serine; Folate and VB12 are the carriers of the one carbon units, trace folate and VB12 could diminish L-serine degradation flux ratio which was directed into glycine and one carbon uint, and increased YP/S and YP/X by accelerating the efficiency of the one carbon units metabolism. The final L-serine productions were 7.1 g/L and 9.7 g/L, which were 39% and 82% higher than the control. These results conform that L-serine is an important intermediate metabolite of C. glutamicum SYPS-062. Moreover, by manipulating these 6 vitamins levels, the distribution of metabolic flux can be discovered and regulated, which is an effective strategy to increase the production of L-serine. Finally, the fermentation period was 6 h shorter by adding all these vitamins at their optimal doses than by adding biotin only, and the accumulation of L-serine and the biomass were 9.0 g/L and 11 g/L, which were 70% and 76% higher than the control.
Effect of organic acids on 1.3 propanediol fermentation was studied in this paper.The adsorption of organic acids from glycerol fermentation liquor by ion-exchange resins was investigated.The results showed that organic acid and 1.3 propanediol production was innegative relationship.The static adsorption showed that ion-exchange resin 005 had the best adsorption abilities of the organic acids in the glycerol fermentation liquor. It was showed that the yield of 1.3propanediol increased by 166% after the extraction of organic acids from glycerol fermentation liquor.
By non-steady method, the effective diffusivity of ferrous sulphate within alginate calcium gel entrapped with bacteria or not. Experimental results showed that, the effective diffusion coefficient of ferrous sulphate decreased with the increase of alginate concentration, the optimum alginate concentration is2%(W/V). The effect of calcium chloride on the effective diffusivity was neglectable. The incubation of thiobacillus ferrooxidans would pass through 10 hours, and the diffusion coefficient within gel entrapped bacteia was less remarkably than that of ferrous sulphate without entranpped bacteria.
A one step RT-PCR was developed to detect H5 subtypes of avian influenza virus (AIV). A pair of specific oligonucleotide primers was designed for the detection of AIV H5 subtypes according to 221 hemagglutinin (HA) gene sequences. Sensitivity to detection of allantoic fluid by one step RT-PCR reached 10 thousand dilutions and detection of swab samples reached 8 dilutions. The tissue and swab samples infected with H5 subtypes AIV were simultaneity detected by one step RT-PCR and virus isolation method. The results showed that the sensitivity of the assay was lower 10 to 100-fold than virus isolation method and the assay gave an excellent (100 %) correlation with the conventional virus isolation method. H1~H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR. The results showed the assays were high specific, without cross-reaction to other subtype or other avian diseases. Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of H5 subtypes AIV.
The main reason of Multidrug resistence is P-gp overexpressed in vivio. Because of its expression and localization, it has been suggested that P-gp plays an important role in cancer chemotherapy. In this study, we performed a method for monitoring P-gp overexpressed. A recombinant plasmid named pETP-gp was constructed by cloning MDR 1 1kb gene into prokaryotic expression vector pET-28b(+). E.coli competent host BL21(DE3) and BL21(DE)lyss were transformed with pETP-gp. The recombinant protein P-gp was high level expression in the two Ecoli BL21(DE3) and BL21(DE)lyss after induced by IPTG. Recombinant proteins were used as detecting antigen, and then the optimal reactive condition and the concentration of the second antibodies were confirmed. The expression in BL21(DE3)plyss was higher until to 48% of the total protein of the induced recombinant bacteria. Expression protein was shown as specificity P-gp by immunoprint. It was the first report that MDR 1 1kb gene product was expressed with high level in E.coli. Indirect ELISA for the detection of serum antibodies against MDR 1 was established with the E.coli high expressed recombinant pETP-gp protein as antigen and the rabbit against human IgG by tagging HRP as the second antibodies.
Culture environment is the key factor in the construction of dermal skin. This study reports on the effects of culture methods on the construction of dermal substitutes using collagen-chitosan sponges in vitro. Through comparing the static culture and spinner flask culture methods, and the spinner flask culture with different stir speeds 10 r/min, 40 r/min and 80 r/min, the effects of the culture environment on the cells proliferation, cells distribution within scaffold and cells metabolism had been studied. A greater cell density was obtained with spinner flask culture versus static culture, especially, the 80 r/min spinner flask culture shows the obvious advantage with the greatest cell density and specific growth rate. The stirred culture of fibroblast-seeded sponges in spinner flasks resulted in a more uniform cell distribution compared to static culture. The dermal substitutes obtained from the 80 r/min spinner flask culture have greater cell density and more uniform distribution within scaffold than the 10 r/min and 40 r/min spinner flaks. In summary, the spinner flasks culture with proper stir speed shows promise for the in vitro construction of dermal substitutes.
Objective: To study the physical and chemical attributes of nanoparticulated aluminum hydroxide adjuvant (NAHA) before and after autoclaving. Methods: Turbimetric Assay (TA), the pH and the diameter of Nanometer-Particles (NP) were applied to this comparison. Results: the pH was slightly decreased during the autoclaving, but there was no turbimetric change and diametric change of the NP. Conclusion： Nanometer-aluminium hydroxide adjuvant can be sterilized by autoclaving during the production of bacterin.
Orange peel was used as low-cost adsorbent in binding of Methylene Blue. The effects of equilibrium time, pH, dye concentration have been studied. Carboxyl, amine and phosphonate functional groups were present in the orange peel. The equilibrium time was 1 hour, the maximum adsorption capacities of the orange peel was 370.3±31.0 mg/g at pH 10. The Langmuir and Freundlich isotherms were well fitted in this biosorption system. Results showed relatively higher rate constant and biosorption capacities. These adsorption performance indicate the orange peel as a potentially economical adsorbent for dye removal.
The influenza A virus matrix protein2 gene(M2) which deleted transmembrane region was amplified by overlap extending PCR, and the multi-epitope gene of hemagglutinin(HA) was PCR amplified with seven continuous synthesized segments by designing primer. The two gene segments were separately cloned into pMD18-T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+-M2d-HA. The recombinant plasmid was transformed into E.coli BL21(DE3), and the high expression strain was obtained by screening monoclones. The recombinant protein existed as inclusion bodies, which accounted about 45% of the total cellular protein. The inclusion bodies were washed with 1% Triton X-100 solution twice, and dissolved in 8 mol/L urea solution. The solution protein was purified by Ni2+ affinity chromatography, and refolded by dilution renaturation, then purified by Q Sepharose FF cation exchange column. The purity of the protein was over 90% by HPLC analysis. The result of western-blot showed it has good antigenicity and specificity. These results strongly supported for the further study of the broad-spectrum influenza virus subunit vaccine.
In order to study the growth behavior of bovine Sertoli cells, adopting the combination of enzymatic digestion and different attaching velocities methods to isolate and purify Sertoli cells of 5-month, 6-month bovine fetal testis and newborn bovine testis, then cultured in vitro. Indicating that bovine testis in this stage were suitable for isolating and purifying Sertoli cells, 0.25% trypsin+0.02%EDTA is an economic and efficient method to isolate bovine fetal Sertoli cells, and the optimal research period of bovine fetal Sertoli cells should be controlled within 3-20 days, bovine fetal Sertoli cells in exponential growth stage can obviously promote the development of bovine IVF zygotes in vitro.
A high-product Superoxide dismutase(SOD) bacterial strain was first obtained from the soil around the warm spring of Changbai Mountain, which is alkaline-resistant. The nature analysis by the inhibition of H2O2 and chloroform-ethanol indicated that the SOD was Mn-SOD. It belongs to bacillus genus by the modal character, physiological and biochemical character. The phylogenetic analyses based on 16SrDNA sequence indicated that the strain 110-2 was closed to Bacillus sp.MO6 with 100% identity and 99% with Bacillus licheniformis. According to the sodA sequence of Bacillus licheniformis and conservative regions of many kinds of bacillus, which were published in GenBank, two pairs of primers were designed and the complete sequence of Mn-SOD(600bp) and the core fragment(430bp) were amplified by PCR techniques. The recombinant plasmid pMD18-SOD was built successfully.
Objective To clone lexA gene, express and purify the repressor LexA from Pseudomonas aeruginosa (PA), prepare the polyclonal antibody against PC-1 protein in rabbits, detect immunological activity of LexA protein. Methods The genomic DNA was extracted from the PAO1, the gene fragment was encoding the mature LexA was amplied by PCR. It was linked the vector pET32a(+) and expressed in the E.coli BL21(DE3).The expressed protein was purified by two steps of Ni2+ chelate affinity chromatography and gel filtration chromatography respectively. The purified LexA protein immune the rabbits by injection and prepare the polyclonal antibody against PC-1 protein. The immunological activity of expressed and purified LexA protein was detected by ELISA , and Western blot. Results The expressed fused protein was found in insoluble form, accounted for 45% of the total bacteria protein. The final purity was 98.97%, which was determined by the HPLC. The expressed and purified LexA protein had satisfactory immunological activity.
CD127 is the interleukin-7 receptor α (IL-7Rα), it regulates the specific respond of T lymphocytes to IL-7. CD127 plays an indispensable role in the development of thymocytes into T lymphocytes, the survival and homeostatic proliferation of memory T cells. There are only two phases of the life period of T cells that do not express CD127: immature CD4+8+double positive (DP) thymocytes and activated T cells. Recently, CD127 was found that it play an important role as the specific marker of memory T cells and regulator T cells. The role and mechanism of CD127 in the life period of T cells were discussed herein
Recently tumor protein D52-like proteins, the first member of which was found in human breast carcinoma, attract increasing research interests. All D52-like proteins are small coil-coil motif bearing proteins which are conserved from lower organism to human being and among the same species. Alternative splicing is producing numbers of isoforms which play different function roles．D52-like genes amplify in multicarcinoma，with the protein expression increasing．The diverse functions of D52-like proteins, which may concern with human disease including cancer, and how they play in vital are not clear so far. Further studies may highlight their function and the mechanism them work.
Muscle recently has been identified as a good source of adult stem cells that can differentiate into cells of different lineages. Researchers have identified two types of stem cells in skeletal muscle. Further research is necessary to delineate the relationship between different populations of muscle-derived stem cells（MDSCs）and between MDSCs and other adult stem cells. The methods used to isolate these cells appear to influence the stem cell characteristics. As these efforts continue, the potential for MDSCs-based therapy for other musculoskeletal injuries, as well as for cardiac and smooth muscle injuries, is currently being explored. This article summarizes the behavior, biocharacteristic, isolation, differentiation and the probability of application to regenerate lost or diseased tissue of MDSCs.
Plant membrane proteomics is one of the most active research fields in plant science. The preparation of plant membrane proteins including plasma membrane, tonoplast and other membrane system for two-dimensional electrophoresis and the proteomics research were reviewed. The database for the proteomics was introduced as well. The prospect of the plant membrane proteomics was raised for reference.
Transgenic plants expressing two or more novel foreign genes have been commercialized and have shown broad perspective. However，one of the major technical hurdles impeding the advance of plant genetic engineering and biotechnology is the fact that the expression or manipulation of multiple genes in plants is still difficult to achieve. A novel expression system of coordinating the expression of multiple protein in a single open reading frame overcome the drawbacks of the conventional techniques which already exist for the stacking of genes and traits in transgenic plants. It is a feasible method for multiple genes transformation. In this article, we discussed the conventional technique introducing multiple genes into plants and the novel polyprotein expression system for transgenic research; and especially described more details about the novel linker peptide 2A and LP4 which were used to connect different protein sequence into one polyprotein.
Polo-like kinase 1(Plk1) play critical roles during multiple stages of cell cycle progression. Plk1 contain an N-terminal Ser/Thr kinase catalytic domain and a C-terminal region that contains two Polo-boxes. Plk1 gene and protein expression has been proposed as a new prognostic marker for many types of malignancies, and Plk1 is a potential target for cancer therapy.
Bioreactor is a key equipment in bioengineering. The bioprocessing efficiency using bioreactors is significantly affected by the reactor configuration. It is apparent that research on the bioreactor structure is one of key issues in bioengineering. Along with industrialization of penicillin mechanically stirred bioreactor was developed, after that large quantity of bioreactors was invented for the sake of the development of mammal and plant cell cultivation ,epiphyte cultivation and algae cultivation. Among these bioreactors mechanical stirred bioreactors and airlift bioreactors are very popular. This article reviews recent trends of studies on mechanically stirred bioreactors and air-lift ones. Our focus is on the summarization and analysis of 11 kinds of novel bioreactors developed around the world.
Recombinant PCR applies to fulfill gene recombination by PCR thermal reaction. Over the twenty years, it has branched into three characteristic strategies: splicing by overlapping extension (SOE), jumping polymerase chain reaction (JPCR) and DNA shuffling. Recently, the technique aimed with exploiting natural source of different allele genes is developing up on simplification of experimental procedure, on trap for mutation and variation, and on high-throughput screening with technology of surface display and fluorescent probe. The recombinant PCR is increasesing value in broad range from biological basic research to bioengineering study.
