pMD18-T作为A组轮状病毒VP7基因克隆载体的研究

Study on cloning vector of pMD18-T containing group A rotavirus VP7 gene

目的:利用RT-PCR法从A组轮状病毒扩增基因片段VP7,再将产物重组于大肠杆菌pMD18-T载体,探讨pMD18-T作为A组轮状病毒VP7基因克隆载体的应用。方法:提取A组轮状病毒总RNA,通过RT-PCR扩增,获得目的基因片段VP7,进行分离纯化和回收,最后把纯化的VP7基因与pMD18-T载体进行连接,进行质粒抽提与鉴定。结果:成功将VP7基因连接到pMD18-T载体,转化感受态菌(DH5α),通过蓝白斑筛选得到DNA阳性重组子(载体+VP7基因片段),经DNA测序鉴定正确。结论:成构建功A组轮状病毒外壳蛋白VP7基因克隆载体,为进一步获得大量VP7基因以及研究和开发轮状病毒基因工程疫苗奠定基础。

Objective：To construct the recombinant pMD18-T vector containing group A Rotavirus VP7 gene by RT-PCR,so as to study on cloning vector of pMD18-T containing group A rotavirus VP7 gene.Methods：Through extracting the total Group A rotavirus RNA and amplifying by RT-PCR,the VP7 gene fragment was isolated and recoveried,and trsansducted into pMD18-T.After VP7 gene was extracted from the plasmid,it was detected by 1% agarose gel electrophoresis.Results：The VP7 gene was transformed into pMD18-T.After screening by blue and white plague,the recombinant DNA （vector and the VP7 gene） was selected;And the sequence analysis results were compared with those in GeneBank.Conclusion：The VP7 gene has been successfully transformed into pMD18-T vector,so as to lay a foundation for the further development of genetic engineering vaccine against rotavirus.