淋球菌主要外膜蛋白PIA原核重组质粒的构建及序列分析

Construction and Sequence Analysis of A Recombinant Plasmid Harbouring the Porin Gene PIA of Neisseria gonorrhoeae

目的构建携带淋球菌主要外膜蛋白PIA基因的pET重组质粒,分析其编码序列。方法根据PIA已知序列设计一对引物,用PCR技术从淋球菌捷克标准株中扩增PIA;将目的基因PCR产物和质粒pET28a(+)同时用BamHⅠ和HindⅢ限制性内切酶进行双酶切,纯化回收后将其连接并将重组质粒转化大肠杆菌DH5α。将构建的重组质粒pET28a(+)-PIA通过PCR、质粒双酶切、DNA测序证实获得正确的重组克隆。结果成功构建PIA基因原核重组质粒pET28a(+)-PIA。序列分析表明,其携带的PIA基因序列与GenBank中公布的淋球菌(AF090818)的PIA序列具有很高的同源性,其同源性达到99.9%。结论成功构建携带淋球菌主要外膜蛋白PIA基因重组表达载体,有助于更深入地分析其基因功能,为后期淋球菌的临床检测和基因疫苗研制打下基础。

Objective To construct a recombinant plasmid harbouring the PIA gene of Neisseria gonorrhoeae. Methods A pair of primers was designed according to the known sequence of PIA gene. The PIA gene fragment was amplified by PCR. After purified and digested with BamHⅠand Hind Ⅲ, the PIA gene fragment was ligated to a prokaryotic expression vector, pET28a(+). Recombinant pET28a(+) PIA was constructed and transformed into E.coli DH5α. Positive recombinant was verified by PCR, restriction mapping and DNA sequencing. Results The recombinant plasmid pET28a(+) PIA was constructed successfully. The result of sequence analysis of the PIA gene in the recombinant plasmid pET28a(+) PIA showed a 99.9%homology with the published sequence in the GenBank. Conclusion The construction of pET28a(+) PIA is important for the further research on the function of PIA and may help in the development of the method for clinical detection and genetic engineering vaccine of Neisseria gonorrhoeae.