生殖支原体MgPa原核载体的构建

Construction of Recombinant Prokaryotic Vector of Mycoplasma Genitalium MgPa

【目的】构建生殖支原体（Mg）黏附蛋白MgPa优势表位基因（MgPa′,1075-1364aa）的原核表达载体。【方法】通过生物信息学分析,筛选并挑选MgPa基因优势抗原表位,以MgG-37标准株基因组DNA为模板,高保真聚合酶链反应扩增目的片段,将其亚克隆到原核表达载体PET-30a（＋）中,构建重组质粒PET-30a（＋）/MgPa＇。【结果】PCR扩增得到大小约为870bp的目的片段;构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片段为MgPa＇目的基因,测序结果与Genbank上登录序列完全一致。【结论】采用上述方法可成功构建生殖支原体MgPa原核载体。

[Objective] To construct recombinant prokaryotic vector containing the gene encoding immunodominant epitope （1075-1364 amino acid residues ）of MgPa attachment protein （MgPa） of Mycoplasma genitalium （M. genitalium）. [Methods] The immuno-dominant epitope of MgPa gene from M. genitalium complete genome was chosen by computer analysis and then was amplified by polymerase chain reactions （PCR）. The PCR product was subcloned into the expression vector PET-30a（＋） to generate recombinant plasmid PET-30a（＋）/MgPa＇. [Results]The size of PCR amplification product was about 870bp. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene was MgPa＇, and it had 100% similarity with gene reported by GenBank. The results of BLAST confirmed that the sequence was MgPg. [Conclusion] Prokaryotic expression vector PET-30a（＋）/MgPa＇was constructed successfully.