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 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 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 a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

Based on a previously used plasmid pHC11, a new plasmid pHC11R was con-structed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

Expression of Avian Reovirus (ARV) σA Protein in HEK293T Cells

[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according to the gene sequence of ARV S2 gene in GenBank(accession number KF741763.1).With pMD18-T-σA recombinant vector as the template,the specific sequence ofσA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant plasmid.The cloning vector pMD18-T-σA and eukaryotic expression plasmid pEF1α-HA were double digested by restriction enzymes Kpn I and Not I.The purifiedσA gene was connected with pEF1α-HA to construct eukaryotic expression plasmid pEF1α-HA-σA.After colony PCR,double enzyme digestion and sequencing,the recombinant plasmid pEF1α-HA-σA was tansfected into HEK293T cells.The proteins were collected at 24 h after tansfection and verified by Western-blot.[Result]The ARVσA gene was successfully cloned in the test.The eukaryotic expression plasmid pEF1α-HA-σA was constructed,which could be expressed in HEK293T cells.[Conclusion]The protein could be accurately expressed in HEK293T cells.

转基因番茄防龋疫苗的基础研究 Ⅰ.变形链球菌pac基因唾液粘附区植物表达质粒的构建

目的 :构建变形链球菌表面蛋白基因 (pac)唾液粘附区的植物表达质粒pROP1和pRPB1。方法 :用PCR扩增的含变形链球菌表面蛋白唾液粘附区P1片段 (184- 1946bp)与植物的高效表达质粒pROKCP结合 ,构建pROP1表达质粒 ;且将此质粒与Bar基因 (抗除草剂基因 )连接 ,构建表达质粒pRPB1,酶切电泳检测。结果 :从pPC41中扩增的P1片段整合到pROKCP的适当部位 ,构成重组质粒pROP1,从pPBar分离出的Bar基因整合到pROP1,构成重组质粒pRPB1。结论 :本实验成功地构建了携带变形链球菌表面蛋白唾液粘附区的植物表达质粒pROP1和pRPB1。

Construction of Three Nucleic Acid Vaccines of Swine Hepatitis E Virus ORF2 Ger\e Continuous Fragment

In order to develop swine hepatitis E （HE） genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.

重组抗菌肽LfcinB/LfampinB基因的克隆及表达载体的构建

根据GeneBank中提供的牛乳铁蛋白基因序列,设计并合成了重组LfcinB/LfampinB基因,并通过PGEM-Teasy载体扩增。酶切回收的LfcinB/LfampinB片段与中间载体pP6连接。pP6上的启动子、信号肽和调控序列与LfcinB/LfampinB共同切下,获得具有上游调控元件的PLfc/Lfa基因片段。将PLfc/Lfa与pME290表达载体连接,并转化绿脓杆菌。结果表明,本试验成功构建了重组pPLfc/Lfa表达载体。