猪瘟病毒Shimen株p80基因的克隆及其真核表达质粒的构建

根据已发表的猪瘟病毒(classicalswinefevervirus,CSFV)Alfort株和Brescia株的全基因组序列,设计2对引物P1/P2和B1/B2,在B1和B2的5''端分别加上XhoI和ApaI位点,以CSFVShimen株细胞毒为材料一步法提取总RNA,并以此为模板采用反转录PCR(RT-PCR)和套式PCR(nPCR),成功地扩增到约2.0kb的片段,将此PCR产物回收后与pMD18-T连接、转化,获得重组质粒,经PCR扩增,限制性酶切(BamhI/HindШ)和序列部分测定鉴定为阳性重组质粒P80-T。将P80-T分别经XhoI和ApaI酶切消化、回收后,与经XhoI/ApaI酶解的真核表达载体PEGPF-C1连接、转化,获得重组质粒,经PCR,XhoI和ApaI限制性酶切和序列测定鉴定为真核表达质粒P80-P,目的基因的插入位置、方向和读码框完全正确。为下一步在哺乳动物细胞中表达猪瘟病毒p80蛋白奠定了基础。

Clone and Eukaryotic Expression Plasmid Construction of p80 Gene of CSFV Strain Shimen

Two pairs of primers P1/P2 and B1/B2 were designed based on the sequence of the upper stream and down stream of CSFV P80 gene. About 2.0kb fragment was reversely transcribed and amplified from CSFV strain Shimen using the primers and further idendified as the p80 gene by gel analysis. The RT-PCR product was purified and cloned into the pMD 18-T Vector. The recombinant plasmid P80-T was indicated containing p80 gene by PCR, restriction enzyme digestion and partial nucleotide sequencing. The P80 gene was subsequently subcloned into expression vector PEGFP-C1 by restriction enzyme Xho I/Apa I digestion and linking,and then the recombinant P80-P with P80 gene was generated, which could be further expressed in eukaryotic cell.