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目的克隆淋病奈瑟菌孔蛋白I B型(PIB)基因并构建其真核表达载体pC I-PIB,了解pC I-PIB免疫接种后诱导小鼠特异性体液和细胞免疫反应的效果。方法采用聚合酶联反应(PCR)扩增淋病奈瑟菌主要外膜蛋白PIB全长基因片段(960 bp),构建表达PIB的真核表达载体pC I-PIB。pC I-PIB肌内注射免疫BALB/c小鼠65只,100μg次//只,亲和素-生物素-过氧化酶复合物(ABC)法检测其中10只pC I-PIB免疫小鼠肌细胞中PIB的表达,酶联免疫吸附试验(ELISA)和特异性淋巴细胞增殖反应(MTT)法检测余下pC I-PIB免疫小鼠的特异性体液和细胞免疫应答效果。采用玻片凝集试验和补体溶菌试验检测pC I-PIB免疫小鼠血清的抗菌活性。结果PCR可扩增出预期大小的全长PIB基因片段(960 bp),与报道PIB基因序列(GenBank No:AF090801)比较,重组质粒pC I-PIB中目的插入片段的核苷酸序列同源性可达99.28%。免疫小鼠的肌细胞能摄取pC I-PIB并表达PIB。pC I-PIB免疫小鼠的血清中可产生较高效价的特异性IgG(1∶4000),并产生特异性T细胞增殖反应,增殖指数(4.031)明显高于对照组(1.127)(t=71.71,P<0.05)。pC I-PIB免疫小鼠血清及阴道冲洗液均有凝集淋病奈瑟菌的作用,在补体参与下可杀灭细菌。结论本研究成功地构建了淋病奈瑟菌PIB基因重组真核表达载体pC I-PIB。pC I-PIB接种小鼠后可有效地引起特异性体液和细胞免疫反应,具有作为淋病奈瑟菌候选DNA疫苗的应用前景。
Objective To clone the gene of porcine Neisseria gonorrhoeae porin I type B (PIB) and construct its eukaryotic expression vector pC I-PIB to investigate the specific humoral and cellular immune responses induced by pC I-PIB inoculation. Methods The full length PIB gene fragment (960 bp) of major outer membrane protein of Neisseria gonorrhoeae was amplified by polymerase chain reaction (PCR), and the eukaryotic expression vector pC I-PIB expressing PIB was constructed. Sixty BALB / c mice were immunized with pC I-PIB and 10 pC I-PIB mice were immunized with 100 μg / ml of avidin-biotin-peroxidase complex (ABC) The expression of PIB, the ELISA and the specific lymphocyte proliferation (MTT) assay were used to detect the specific humoral and cellular immune response in mice immunized with pC I-PIB. The antibacterial activity of pC I-PIB immunized mice serum was tested by slide agglutination test and complement lysis test. Results PCR amplified the full-length PIB gene fragment (960 bp) with the expected size. Compared with the reported PIB gene sequence (GenBank No: AF090801), the nucleotide sequence homology of the inserted fragment in the recombinant plasmid pC I-PIB Up to 99.28%. Muscle cells from immunized mice were able to uptake pC I-PIB and express PIB. The pC I-PIB immunized mice could produce higher titers of specific IgG (1: 4000) and produce specific T cell proliferation response (4.031) than the control group (1.127) (t = 71.71, P <0.05). pC I-PIB immunized mice serum and vaginal washings have the role of agglutination Neisseria gonorrhoeae, with complement can kill bacteria. Conclusion This study successfully constructed a recombinant eukaryotic expression vector pC I-PIB of Neisseria gonorrhoeae PIB gene. The pC I-PIB inoculated mice can effectively induce specific humoral and cellular immune responses, which has the potential application as a candidate DNA vaccine for Neisseria gonorrhoeae.