摘要
目的研究淋病奈瑟球菌(NG)表面Porin B(Por B)蛋白特异性及免疫保护性在临床中的应用价值。方法登录Gen Bank获取NG世界卫生组织(WHO)标准株E株Por B-PIA基因,设计引物与合成,培养WHO标准株E株,通过聚合酶链反应(PCR)扩增出Por B-PIA DNA片段,采用GST·Bind^(TM)树脂纯化Por B蛋白,对噬菌体随机12肽库进行筛选并对阳性克隆进行提取,应用DNA测序及MIXOX软件分析;将培养后的人尿道上皮细胞分别与Por B蛋白特异性结合的多肽、NG活菌进行培养,并将未加入抗体作为对照组。观察Por B蛋白的纯化表达,Western blot检测Por B蛋白与NG特异性关系,间接酶联免疫吸附试验(ELISA)检测筛选后蛋白的特异性抗体水平,比较Por B蛋白特异性结合多肽对细胞内形成微菌落的差异。结果Ecoli-BL21中克隆表达的诱导产物经Ni+-NTA Purification System纯化后,获得纯度较高的Por B蛋白;制备出Por B蛋白相应的特异性结合多肽,蛋白出现特异性条带;间接ELISA检测筛选后蛋白的特异性抗体水平,筛选后蛋白的抗体水平在1∶200;加入Por B蛋白特异性结合多肽的组形成的微菌落显著少于对照组(P<0.05)。结论NG表面Por B蛋白发挥显著特异性和免疫保护性,但还需大量研究及临床试验来证明其临床效果并进一步分析Por B蛋白的相关机制和作用,为临床NG疫苗的研究提供依据。
Objective To study the clinical application value of the specificity and immunoprotection of Porin B(Por B)protein on the surface of Neisseria gonorrhoeae(NG).Methods The Por B-PIA gene of the world health organization(WHO)standard strain E of NG was obtained from Gen Bank,primers were designed and synthesized,and the standard strain E of the WHO strain was cultured.The Por B-PIA DNA fragment was amplified by PCR,and the Por B protein was purified by GST·Bind^(TM) resin.The phage random 12 peptide library was screened and positive clones were extracted,DNA sequencing and MIXOX software analysis were used;the cultured human urethral epithelial cells were cultured with peptides specifically binding to Por B protein and NG live bacteria,and no antibody was added as a control group.The purified expression of Por B protein was observed.The specific relationship between Por B protein and NG was identified by Western blot.Indirect enzyme-linked immunosorbent assay(ELISA)was used to detect and screen the specific antibody level of the protein.The difference of Por B protein specific binding polypeptide on the formation of microcolonies in cells was compared.Results After the induced product cloned and expressed in Ecoli-BL21 was purified by the Ni+-NTA Purification System,the Por B protein with higher purity is obtained.The specific binding peptide corresponding to the Por B protein was prepared,and the protein has a specific band.After the specific antibody level of the protein is screened by indirect ELISA,the antibody level was 1∶200.The microcolonies formed by the Por B protein-specific binding polypeptide group were significantly lower than those of the control group(P<0.05).Conclusions The Por B protein on the surface of NG exerts remarkable specificity and immunoprotection,but a large number of studies and clinical trials are needed to prove its clinical effect and further analyze the related mechanism and role of Por B protein,so as to provide a basis for clinical NG vaccine research.
作者
吴学良
戴礼记
王威
李兴
张妮
易军
曾志平
刘拥荣
WU Xueliang;DAI Liji;WANG Wei;LI Xing;ZHANG Ni;YI Jun;ZENG Zhiping;LIU Yongrong(Andrology Center,Ningxiang Hospital Affiliated to Hunan University of Traditional Chinese Medicine,Ningxiang 410600,Hunan,China;Department of Pharmacy,Ningxiang Hospital Affiliated to Hunan University of Traditional Chinese Medicine,Ningxiang 410600,Hunan,China;Medical Laboratory Center,Ningxiang Hospital Affiliated to Hunan University of Traditional Chinese Medicine,Ningxiang 410600,Hunan,China)
出处
《中国性科学》
2022年第8期136-140,共5页
Chinese Journal of Human Sexuality
基金
湖南省卫生计生委科研计划课题(B2016223)
宁乡市人民医院校院联合科研基金项目(2019LLXY10)。
