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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 predominant distribution of porin Ⅰ gene in clinical isolates of Neisseria gonorrhoeae in Zhejiang province and construct the prokaryotic expression system of porin Ⅰ A and porin Ⅰ B genes. Methods Based on the previous studies, the nucleotide sequences of porinⅠA and porinⅠB were determined by T-A cloning. The dominant genotypes of porinⅠA and porinⅠB were analyzed. The prokaryotic expression system of porinⅠA and porinⅠB genes was reconstructed. After induced by 0.5 mmo L / L IPTG, the protein expression was detected by 10% SDS-PAGE. Results All the 5 porinⅠA isolates sequenced were serotype ⅠA-6. Among the 11 porinⅠB sequenced strains, 5 serotypes ⅠB-3, 3 serotypes ⅠB-3/6, 1 serotype ⅠB-6, The remaining two strains were mutants. The expression level of PⅠA in the prokaryotic expression system PET-42-PⅠA was 30% of the total bacterial biomass. The expression level of PⅠB in PET-42-PⅠB was 20% of the total bacterial biomass. Conclusion The clinical isolates of porcine Neisseria gonorrhoeae from Zhejiang province are predominant serotype ⅠA-6, porin ⅠB is predominant serotype ⅠB-3, porinⅠA is more conserved than porinⅠB. The constructed prokaryotic expression system can express PⅠA and PⅠB recombinant proteins efficiently.