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目的构建共表达羊布氏菌胞质结合蛋白P39基因和增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的逆转录病毒质粒。方法提取羊布氏菌全基因组DNA,PCR扩增P39基因,连接至pMD19-T载体中,转化感受态大肠杆菌DH5α,经克隆筛选并进行序列测定后,将P39基因连接至逆转录病毒载体pRevIRES-EGFP的多克隆位点上,转化感受态大肠杆菌DH5α,提取重组逆转录病毒质粒pRevP39-IRES-EGFP,进行酶切鉴定。结果从羊布氏菌基因组中扩增出约1 400 bp的P39基因,与GenBank中登录的羊布氏菌16M菌株P39基因序列同源性达99%;重组逆转录病毒质粒pRevP39-IRES-EGFP经双酶切鉴定构建正确。结论已成功构建了共表达羊布氏菌P39基因和EGFP基因的重组逆转录病毒质粒pRevP39-IRES-EGFP,为后续研究奠定了基础。
Objective To construct a retroviral plasmid coexpressing the P39 gene and the enhanced green fluorescent protein (EGFP) gene of B. lamblia. Methods P39 gene was amplified by PCR and ligated into pMD19-T vector and transformed into competent E. coli DH5α. After cloning and sequencing, P39 gene was ligated into retroviral vector pRevIRES -EGFP multi-cloning site, transformed competent E. coli DH5α, recombinant retroviral plasmid pRevP39-IRES-EGFP was extracted, and identified by restriction enzyme digestion. Results The P39 gene of about 1 400 bp was amplified from the genome of M. brevis, and the homology was 99% with the P39 gene of 16M strain of M. lamblia in GenBank. The recombinant retrovirus plasmid pRevP39-IRES-EGFP Identified by double enzyme construction is correct. Conclusion The recombinant retrovirus plasmid pRevP39-IRES-EGFP co-expressing P39 gene and EGFP gene of B. pastoris has been successfully constructed, which lays the foundation for further research.