| | | | | 淋病奈瑟菌主要外膜蛋白DNA疫苗的构建及免疫效果观察 | | | 廖芳;贺超;刘海鹏;宋启发;严杰 | | | 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。
【作者单位】：华中科技大学同济医学院病原生物学系;华中科技大学同济医学院病原生物学系;华中科技大学同济医学院病原生物学系;华中科技大学同济医学院病原生物学系;浙江大学医学院病原生物学教研室 430030武汉;430030武汉;430030武汉;430030武汉
【关键词】：奈瑟氏球菌,淋病;细菌外膜蛋白质类;疫苗,DNA;免疫性
【基金】：湖北省卫生厅资助项目(No.JX1B037)
【分类号】：R759.2
【DOI】：CNKI:ISSN:0253-9624.0.2006-06-011
【正文快照】：
接种疫苗可能是控制淋病流行的有效措施之一,但至今尚无淋病疫苗上市。曾经有学者用淋病奈瑟菌全菌死疫苗免疫人和动物[1],发现对人无任何保护作用,且毒性较大;而该菌菌毛疫苗的临床试验又失败[2],致使淋病疫苗的研究长期停滞不前。直到2004年Zhu等[3]报道,将构建的淋病奈瑟菌主要外膜蛋白DNA疫苗通过肌内注射和基因枪免疫动物产生较明显的免疫反应,才使淋病疫苗的研究有了进一步的发展,但是他们的实验结果有些缺陷,即产生的抗体体外无显著的杀灭淋病奈瑟菌的作用。表达淋病奈瑟菌膜蛋白的DNA疫苗的优势在于可以避免全菌免疫所产生的抗R… | | |  推荐 CAJ下载  PDF下载 | | | CAJViewer7.0阅读器支持所有CNKI文件格式，AdobeReader仅支持PDF格式 | | | | Construction of the DNA vaccine of major outer membrane protein of Neisseria gonorrhoeae and investigation of immune effects after vaccination | | | LIAO Fang~*;HE Chao;LIU Hai-peng;SONG Qi-fa;YAN Jie.*Department of Microbiology;School of Basic Medical Sciences;Tongji Medical College;Huazhong University of Science and Technology;Wuhan 430030;China | | | 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 complement 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.
【Keyword】：Neisseria gonorrhoeae;Bacterial outer membrane proteins;Vaccines,DNA;Immunity |
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