摘要
目的：利用反转录病毒载体构建猪载脂蛋白B mRNA编辑酶催化多肽样蛋白（APOBEC）3F重组质粒,并实现其在猪肾细胞PK15中的表达。方法：用RT-PCR方法扩增五指山猪来源的外周血淋巴细胞APOBEC3F基因,将其定点插入反转录病毒载体pMSCV neo中,同时于插入位点两侧分别添加FLAG和GFP标签,构建重组质粒pMSCV-FLAG-A3F-GFP,并进行酶切、测序鉴定;将鉴定正确的重组质粒与pVSV-G、pGag-Pol共转染包装细胞HEK293T,分别于转染后48~72 h收集细胞的培养上清以获得假型病毒粒子;用该假型病毒感染猪源细胞PK15,通过PCR、Western印迹检测目的基因的整合及表达。结果：PCR扩增到1254 bp的猪APOBEC3F基因,重组质粒pMSCV-FLAG-A3F-GFP经酶切、测序,结果无误;3质粒共转染HEK293T细胞包装出的假型病毒感染PK15细胞后观察到GFP表达;从感染假型病毒的PK15细胞基因组中扩增到1254 bp的猪APOBEC3F基因,Western印迹检测到78.1×103的猪APOBEC3F蛋白的表达。结论：实现了反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的整合与表达,为深入研究该分子对猪内源性反转录病毒（PERV）的抑制作用奠定了基础。
Objective： To construct an eukaryotic expression plasmid with porcine apolipoprotein B mRNA-edit ing,enzyme-catalytic polypeptide-like（APOBEC） 3F gene and transfect into swine PK15 cell line by retroviral vec tor system.Methods： The APOBEC3F gene was amplified by RT-PCR from the peripheral blood mononuclear cells of Wuzhishan miniature pigs,and then the APOBEC3F gene was inserted into retroviral vector pMSCV neo to construct a recombinant plasmid pMSCV-A3F-FLAG-GFP with adding the two end-tags FLAG and GFP.The plasmid was identified by enzyme digesting and sequencing analysis.The plasmid,together with pVSV-G and pGag-Pol,was co-transfected into HEK293T packaging cells to prepare the pseudotype virus.PK15 cells were then transfected by the viron particles,which were the harvesting of supernatant of HEK293T packaging cells after 48~72 h.PCR and Western blot were applied to identify to integration and expression of porcine APOBEC3F gene.Results： Length of 1254 bp of porcine APOBEC3F gene was amplified and the recombinant plasmid pM SCV-A3F-FLAG-GFP was identified correctly by enzyme digesting and sequencing analysis.GFP expression was observed in PK15 cells after the transfection of HEK293T cells with three plasmids.PCR results showed that 1254 bp of procine APOBEC3F gene could be detected in the PK15 cell genomic DNA after infecting with the pseudotype virus.Western blot results showed the protein expression of 78.1 kDa procine APOBEC3F.Conclusion： Porcine APOBEC3F gene mediated by retroviral vector system can be integrated and expressed in PK15 cells.Our results provided the support for the research of inhibition of porcine endogenous retrovius by porcine APOBEC3F.
出处
《生物技术通讯》
CAS
2012年第5期686-689,共4页
Letters in Biotechnology
基金
国家自然科学基金(31071985
30800829)
北京市自然科学基金(6092020)
