猪NMB基因过表达载体的构建及其在猪睾丸间质细胞中的表达

Construction of porcine NMB gene overexpression vector and its expression in porcine leydig cells

为了构建猪神经介素B(pNMB)基因慢病毒过表达载体,以及研究其在猪睾丸间质细胞中稳定表达情况,试验采用RT-PCR方法扩增获得pNMB基因的CDS序列,插入到pMD-19T载体中,构建pMD19-T-pNMB重组质粒,对该重组质粒和慢病毒过表达质粒pCD513B-1进行双酶切,以获得的线性化pCD513B-1和线性化pNMB质粒构建pCD513B-1-pNMB重组慢病毒过表达载体;将重组慢病毒过表达载体pCD513B-1-pNMB和辅助质粒(pGag/Pol、pRev和pVSV-G)共转染至HEK293T细胞中,包装获得pNMB过表达慢病毒,并用倍比稀释法测定病毒含量;用pNMB过表达慢病毒转染猪睾丸间质细胞,通过嘌呤霉素抗性筛选获得稳定表达的细胞,利用实时荧光定量PCR方法检测pNMB mRNA的表达情况。结果表明:RT-PCR扩增获得大小约为366 bp的目的片段,与GenBank中pNMB基因的CDS序列完全一致,RT-PCR扩增片段与目的片段序列完全相同;将pNMB基因克隆到pMD-19T载体中,成功构建出pMD19-T-pNMB重组质粒;XbaⅠ和EcoRⅠ双酶切重组质粒pMD19-T-pNMB和慢病毒过表达载体pCD513B-1后获得线性化pCD513B-1和线性化pNMB,并成功构建了重组慢病毒过表达载体pCD513B-1-pNMB;包装获得了pNMB过表达慢病毒,病毒含量约为5×10^(6) TU/mL;pNMB过表达慢病毒成功转染了猪睾丸间质细胞,筛选获得pNMB mRNA稳定高表达的细胞。说明构建的pNMB过表达慢病毒可用于研究过表达pNMB基因对猪睾丸间质细胞的影响。

In order to construct porcine ganglionin B(pNMB)gene lentiviral overexpression vector and to study its stable expression in porcine Leydig cells,in the experiment,the CDS sequence of the pNMB gene was amplified by RT-PCR and inserted into the pMD-19 T vector to construct the pMD19-T-pNMB recombinant plasmid.The recombinant plasmid and the lentiviral overexpression plasmid pCD513 B-1 were double digested to obtain the linearized pCD513 B-1 and linearized pNMB plasmids to construct the pCD513 B-1-pNMB recombinant lentiviral overexpression vector.The recombinant plasmid pCD513 B-1-pNMB and helper plasmids(pGag/Pol,pRev and pVSV-G)were co-transfected into HEK293 T cells to obtain pNMB overexpression lentivirus,and the virus titer was measured by double dilution method.Porcine Leydig cells were transfected with pNMB overexpressing lentivirus,and stably expressed cells were obtained by puromycin resistance screening.The expression of pNMB mRNA was detected by real-time fluorescent quantitative PCR.The results showed that the target fragment with a size of about 366 bp was obtained by RT-PCR amplification,which was completely consistent with the CDS sequence of the pNMB gene in GenBank.The pNMB gene was cloned into the pMD-19 T vector,and the RT-PCR amplified fragment was exactly the same as the target fragment,and the pMD19-T-pNMB recombinant plasmid was successfully constructed.The recombinant plasmid pMD19-T-pNMB and the lentiviral overexpression vector pCD513 B-1 were digested with XbaⅠand EcoRⅠto obtain linearized pCD513 B-1 and linearized pNMB;and the recombinant lentiviral overexpression vector pCD513 B-1-pNMB was successfully constructed.The pNMB overexpression lentivirus was obtained by packaging,and the virus titer was about 5×10^(6) TU/mL.The pNMB overexpression lentivirus was successfully transfected into porcine Leydig cells,and the cells with stable and high expression of pNMB mRNA were obtained.The results suggested that the constructed pNMB lentiviral overexpression vector could be used to study the effect of overexpression of NMB gene on porcine Leydig cells.