淋病奈瑟菌主要外膜蛋白DNA疫苗的构建及免疫效果观察

Construction of the DNA vaccine of major outer membrane protein of Neisseria gonorrhoeae and investigation of immune effects after vaccination

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 PIB gene of Neisseria gonorrhoeae, and to construct a recombinant eukaryotic expression vector pCI-PIB and to understand the effects of pCI-PIB vaccination in mice to induce specific humoral and cellular immune responses. Methods The entire PIB gene of Neisseria gonorrhoeae （960 bp） was amplified by using PCR. An eukaryotic eukaryotic vector pCI-PIB was then constructed. BALB/c mice （n = 65, 100 μg/time/mouse） were immunized with pCI-PIB by intramuscular injection. ABC assay was employed to examine the PIB expression in muscular cells of the pCI-PIB-immunized mice （n=10）. ELISA and MTT assays were used to measure the effects of humoral and cellular immune responses of the remaining pCI-PIB-immunized mice. By using slide agglutination test and compleraent bacteriolytic test, the serum anti-bacterial activity of the pCI-PIB immunized mice was determined. Results The entire PIB gene amplification fragment of the expected size （960 bp） was successfully obtained by PCR. In comparison with the reported PIB gene sequence （GenBank No： AF090801）, the homology of nucleotide sequence of the target inserted fragment in the recombinant plasmid pCI-PIB was as high as 99.28%. The muscular cells of the immunized mice could take in pCI-PIB and then express PIB. In the pCI-PIB immunized mice, the higher titer （1 ： 4000） of specific serum IgG and the specific T lymphocyte response were found. The proliferation index （4.031） was significantly higher than that of the controls （1.127） （t = 71.71, P 〈 0.05）. The sera and washings from the pCI-PIB immunized mice could agglutinate Neisseria gonorrhoeae and kill this microbe in presence of complements. Conclusion In this study we successfully constructed a recombinant eukaryotic expression vector pCI-PIB. The mice inoculated with pCI-PIB might efficiently produce the specific humoral and cellular immune responses, suggesting that pCI-PIB should be potential service as a candidate of Neisseria gonorrhoeae DNA vaccines.