Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

Construction of Shuttle Expression Plasmid and Stable Expression of Foreign Gene in Mycobacteria and E．Coli

By employing the pUC19 as a backbone,the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid PBCG-8000 was constructed. The PBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.

Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells

After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E.coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.

Construction and Expression of Recombinant Plasmid pCD-rbFGF in Osteoblasts

Summary: To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA 3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration(hALR), Expression and Purification of hALR

Experimental evidence has been presented to suggest that the human augmenter of liver regeneration （hALR） serves as a hepatotruphic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by means of Western Blotting. The protein was purified by Ni-NTA affinity chrumatography and this FAD-dependent sulthydryl oxidase activity was measured.

Construction of plasmid containing human blood- clotting factor Ⅷ and observation its expression in Cos-7 cells

Factor Ⅷ deficiency or hemophilia A is X-linkedgenetic disorder in human. General treatment of severehemophilia A consists of adiministration of plasma-derived or recombinant clotting factor concentrates. Ithas caused a series of problems, i. e. viral infection andcost too much to use rF Ⅷ. Nowadays, people havedeveloped the retroviral vector and the adcnoviral

Construction and Expression of Recombinant Ghrelin Plasmid and Effects on Growth Performance and Gastric Acid Secretion of Rats

The paper was to study construction and expression of rGhrelin （pcDNA3 -Ghrelin） and its effect on growth performance and gastric acid secretion of rats. Ghrelin amplified from gastric mucosa of weaned piglets was cloned into the expression vector pcDNA3 to get recombinant plasmid pcDNA3 - Ghrelin. Twelve weaning rats were randomly divided into two groups, six rats each group. The rats in each treatment group were individually injected with 100pg of naked plasmid pcDNA3 -Ghrelin, and the rats in control group were injected with empty plasmid. The weights and feed consumption of rats were measured after injection for 7, 14 and 29 d, respectively. The rats were sacrificed at the end of the experiment, and their stomach was separated and weighed, the pH value of gastric juice was measured as well. The results showed that the average daily gain of rats at 7 and 29 d were significantly higher than that in control group, respectively （P〈0.05）, and feed consumption did not have significant chan- ges; the feed meat ratio of rats in the treatment groups was significantly lower than that in control group （ P 〈0.05） ; the gastric relative weight and gastric weight did not change significantly, while the pH value of gastric juice of rats in treatment groups was significantly lower than that in control group （P 〈 0.05）. This indicated that after transfected expression of muscle tissue, ghrelin played an important regulatory role in growth and gastric acid secretion of rats.

Construction and expression of GFP conjugated MIM-I-BAR

To achieve a visible inverse Bin-amphiphysin-Rvs （I-BAR）domain recombinant of missing in metastasis （MIM） protein,the green fluorescent protein （GFP）encoding gene was cloned at the terminal of MIM-I-BAR as a probe.The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid.The non-GFP labeled MIM-I-BAR encoding plasmid was also constructed as a control. Being successfully transformed into BL21 （DE3 ）cells,the GFP-conjugated MIM-I-BAR （MIM-I-BAR-GFP ） exhibits strong visible fluorescence,and the expression product can be easily detected by visual inspection, a fluorescence microscope, Western blot or ultraviolet and visible spectrophotometer. Moreover, examination of expression efficiency under various culture conditions revealed that the MIM-I-BAR-GFP gene has a high protein yield at 10 ℃,but not at the culture temperature of 37 ℃.This property is much different from that of the non-fluorescent MIM-I-BAR gene. This optimal expression condition is also proved to be feasible for protein production in midi-scale. The fluorescent recombinant MIM-I-BAR-GFP protein can serve as a useful tool in scientific research, biomedical application and pharmaceutical development.

Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917

The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.

Expression of Foreign Gene in Mycobacterium Regulated by Human Mycobacterium Tuberculosis Heat Shock Protein 70 Promoter

The DNA fragments of 150bp length promoter 0f human Mycobacterium(M.) tuberculosis heat shock protein (hsp)7O and 650bp length foreign gene, the Schistosoma japonicum glutathione S-transferase(Sj26GST)gene,were obtained by amplification with polymerase chain reaction. And the 150p DNA sequence upstream initiation codon ATG of the human M. tuberculosis hsp7O promoter that contains the sequence TTGAG and ATCATA which consensus with E. coli promoter's -35 and-10 region respectively, as well as ribosome binding site GGAGG at position-12-8 upstream the ATG were determined by SangerDideoxyribonucleotide-mediated chain-termination method-Then, the human M. tuberculosis hsp70 promoter and Sj26GST cDNA were cloned into E. coli-mycobacteria shuttle plasmid pBCG-2000 to construct E. coli-Mycobacterium expression shuttle plasmid pBCG- Sj26 that can express Sj26GST gene.The M. smegmatis were electroporated and the positivecolonies were selected by kanamycin-The M.smegmatis containing the vector pBCG-Sj26 can be induced by heating and hydrogen peroxide (H2O2) to express GST. The molecular weight of the recombinant GST(rGST) was 26000. The rGST contents that were about 10 percent of the total bacterial protein were analyzed by density scanning after running SDS-PAGE. This study would provide scientific evidences for application of hsp70 promoter in expressing foreign gene in mycobacterium and development of mycobacterium as multiple-valent vectoral vaccine.

High expression of human augmenter of liver regeneration in E.coli

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Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Stable Surface Expression of a Gene for Helicobacter pylori Toxic Porin Protein with pBAD Expression System

Helicobacterpylori （1f. pylori） infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its conservative nature among 11. pylori, and for its potential as a vaccine candidate. To achieve stable surface expression of this host cell-toxic protein, hopE gene was introduced into pBAD expression system. After induction with arabinose, all 15 randomly-chosen E. coli LMG 194 colonies from 3 successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indirect immunofluorescence confirmed the expression of HopE on E. coli cell surface

Expression,purification and immunocharacteristics of recombination UreB protein of H.pylori

INTRODUCTIONHelicobacter pylori (H . pylori) is associated with the development of chronic gastritis ,peptic ulcer and gastric cancer and gastric MALT lymphoma[1-9],H .pylori has many antigens ,including urease ,heat shock protein and vacuolating cytotoxin and so on ,and urease is an important factor in the colinization of the gastric mucosa and suspected to cause damage to the gastric mucosa[10-14].At the same time ,urdase is also one of the important protective antigens .

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Construction of heat shock protein 90α expressing plasmid and the effect of the protein on tumor growth in vivo

Heat shock protein 90 (Hsp90) is a major heatshock protein whose functions are necessary for livingorganisms, including both procaryotes and eucaryotes.Human Hsp90 consists of two forms, Hsp90α andHsp90β. Expression of Hsp90α appears to be inducible bydifferent stimuli. Adenovirus EIA gene products andtransformation with the H-ras oncogene can induce aselective overexpression of Hsp90α. Hsp90α was