禽脑脊髓炎病毒VP1基因植物表达载体的构建

The Construction of Plant Expression Vector of AEV VP1 Gene

以提取的禽脑脊髓炎病毒总RNA为模板,以P1和P2为引物,反转录为cDNA,再以cDNA为模板进行PCR扩增。将扩增产物克隆pMD18-T载体中,获得重组质粒pMD18-T-VP1,PstⅠ/BamHⅠ双酶切及PCR确认正确后,利用pMD18-T-VP1重组质粒为模板,以P3和P4为引物,经PCR扩增获得了大小约810bp的产物。VP1基因和pBI121植物表达载体用SmaⅠ和SacⅠ双酶切,经纯化后连接,再转化到EHA105农杆菌中,酶切和测序鉴定结果表明,成功地构建了重组质粒pBI121-VP1,为进一步利用转基因植物生产VP1蛋白奠定了基础。

Using P1 and P2 as primers and extracted Avian encephalomyelitis virus total as template,810 bp VP1 gene fragment was amplified by RT-PCR. VP1 gene fragment was cloned into pMD18-T vector,The positive plasmid contained VP1 gene was determinded by restriction analysis and PCR. Then using P3 and P4 as primers and plasmid pMD18-T-VP1 as template,VP1 gene fragment was obtained by PCR amplication. then,VP1 gene and plant expression vector pBI121 were digested with Sma I and Sac I,extracted,connected,recombinant plasmid was transformed into agrobacterium tumefaciens EHA105 by freeze-thawing method,recombinant plasmid pBI121-VP1 was successfully constructed by digested and sequenced.