淋病奈瑟菌外膜蛋白PorB的克隆表达与鉴定

目的　构建表达淋病奈瑟菌外膜蛋白PorB重组质粒 ,并在大肠杆菌中表达获得基因重组蛋白。方法　用PCR方法从淋病奈瑟菌WHO标准株E株基因组扩增出PorB基因片段 ,插入 pUCm T载体中 ,序列测定正确后 ,将其亚克隆到表达载体 pQE3 0中 ,进一步测序正确后在大肠杆菌M 15中表达。 结果　获得淋病奈瑟菌PorB蛋白基因 ,经序列测定与GenBank公布的序列同源性高达 99% ;表达蛋白经SDS PAGE分析 ,相对分子质量与预期大小一致 ;重组蛋白经Western印迹法鉴定 ,在 3 4kD位置有特异条带 ,PorB蛋白在宿主菌中高表达。结论　基因重组表达的融合蛋白将有利于进一步研究淋病奈瑟菌外膜蛋白PorB的功能 ,并有可能用于淋病预防疫苗的研制

Cloning, Expression and Identification of Porin PorB of Neisseria Gonorrhoeae

Objective To construct the recombinant plasmid of porin PorB of Neisseria gonorrhoeae and to express fusion protein in E.coli M15.Methods The gene encoding PorB protein was amplified by polymerase chain reaction (PCR) from genome of Neisseria gonorrhoeae WHO E strain and inserted into cloning vector pUCm-T to form pUCm-T/PorB recombinants.After sequencing,the gene was subcloned to the expression vector pQE30.Sequencing again,recombinant PorB was expressed in E.coli M15.Results The DNA sequence of PorB had 99% homogeneity with that published by GenBank.The anticipate molecular weight(MW) of the protein corresponded to the MW shown in SDS-PAGE.Western blot proved that PorB fusion protein with a molecular weight of 34kD was highly expreessed in E.coli M15.Conclusion The recombinant protein which was constructed and highly expressed in E.coli will assist in the functional characterization of PorB and may be a potential candidate to be used as a vaccine for Neisseria gonorrhoeae.