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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 amplify the gene fragment VP7 from group A rotavirus by RT-PCR and then recombine the product in E. coli pMD18-T vector to investigate the application of pMD18-T as a group A rotavirus VP7 gene cloning vector. Methods: Total RNA of group A rotavirus was extracted and amplified by RT-PCR. The target gene fragment VP7 was obtained and isolated and purified. The recombinant VP7 gene was ligated with pMD18-T vector for plasmid extraction and identification . Results: VP7 gene was successfully ligated into pMD18-T vector and transformed into competent cells (DH5α). DNA positive recombinant (vector + VP7 gene fragment) was screened by blue-white screening. Conclusion: The construction of a set of rotavirus coat protein VP7 gene cloning vector, in order to obtain a large number of VP7 gene and lay the foundation for the research and development of rotavirus gene engineering vaccine.