浙江地区奈瑟淋球菌临床菌株porinⅠ基因优势型及原核表达系统的构建

The dominant genotypes of porinⅠ gene of Neisseria gonorrhoeae isolates in Zhejiang Province and the construction of the prokaryotic expression system

目的分析浙江地区奈瑟淋球菌临床菌株porinⅠ基因优势型分布情况,构建porinⅠA与porinⅠB基因的原核表达系统。方法在先前研究的基础上,对确定的porinⅠA和porinⅠB基因进行T-A克隆后测定核苷酸顺序,分析porinⅠA和porinⅠB的地区优势基因型。再构建porinⅠA和porinⅠB基因优势基因型的原核表达系统,0.5 mmo L/L IPTG诱导后,10%SDS-PAGE检测蛋白表达情况。结果测序的5株porinⅠA全为血清型ⅠA-6;测序的11株porinⅠB中,血清型ⅠB-3有5株,血清型ⅠB-3/6有3株,血清型ⅠB-6有1株,其余2株为变异株。构建的原核表达系统PET-42-PⅠA的PⅠA表达量占细菌总蛋白量的30%;PET-42-PⅠB的PⅠB表达量占细菌总蛋白量的20%。结论浙江地区奈瑟淋球菌临床菌株porinⅠA以血清型ⅠA-6为优势基因型,porinⅠB以血清型ⅠB-3为优势基因型,porinⅠA相对于porinⅠB而言更加保守。所构建的原核表达系统能高效表达PⅠA与PⅠB重组蛋白。

Objective To analyze the dominant genotypes of porin Ⅰ gene encoding major outer membrane protein of Neisseria gonorrhoeae in Zhejiang Province and to construct the prokaryotic expression systems of porinⅠA and porinⅠB genes.Methods Based on the previous research,the porinⅠA and porinⅠB genes were sequenced after T -A cloning, and the dominant genotypes of porinⅠA and porinⅠB genes were analyzed.Then the prokaryotic expression systems of the dominant genotypes of porinⅠA and porinⅠB genes were constructed.Ten percent SDS -PAGE was applied to measure the output of PⅠA and PⅠB proteins after inducement by 0.5 mmol/L IPTG.Results All 5 porinⅠA isolates sequenced were serovar ⅠA -6.Of the 11 porinⅠB isolates sequenced,there were 5 serovarⅠB -3 isolates,3 serovarⅠB -3 /6 isolates,1 serovarⅠ B -6 isolate and 2 mutant isolates.Outputs of P Ⅰ A and P Ⅰ B expressed by the constructed prokaryotic expression systems PET -42 -PⅠA and PET -42 -PⅠB were as high as 30% and 20% respectively. Conclusion ⅠA -6 is the dominant genotypes of porinⅠA gene and ⅠB -3 is the dominant genotypes of porinⅠB gene in Zhejiang Province.Comparing to the porinⅠB gene,porinⅠA gene is more conserved.The prokaryotic expression systems with high efficiency of porinⅠA and porinⅠB genes were successfully constructed,which may be helpful in the further research of genetic engineering vaccine and clinical detection of Neisseria gonorrhoeae.