【摘要】 根据已发表的猪瘟病毒(classicalswinefevervirus,CSFV)Shimen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EcoR和BamH位点,以CSFV-Shimen株脾毒为材料,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),成功地扩增出约1.2kb的E2基因。将PCR产物回收后与pMD18-T载体连接并转化,经PCR和酶切鉴定获得重组质粒E2-T。将E2-T经EcoR和BamH酶切消化、回收目的基因后,与经BamH/EcoR酶切的逆转录病毒载体pBABE-puro连接并转化,经酶切鉴定获得重组质粒pBABE-puro-E2。序列测定表明,目的基因的插入位置、方向和读码框完全正确。更多还原

【Abstract】 Two pairs of primers, P1/P2 and B1/B2, were designed based on the sequence of the upper stream and down stream of CSFV E2 gene. About 1.2 kb fragment was reversely transcribed and amplified from CSFV Shimen strain using the primers and further identified as the E2 gene by gel analysis. The RT-PCR product was purified and cloned into the pMD 18-T Vector. Their recombinant plasmid E2-T was indicated containing E2 gene by PCR and restriction enzyme digestion. The E2 gene was subsequently subcloned into expression vector pBABE-puro by restriction enzyme BamHⅠ/EcoR I digestion and linking. The recombinant pBABE-puro-E2 was generated, which could be further expressed in eukaryotic cell.更多还原