淋病奈瑟球菌表面蛋白A基因疫苗的构建及其诱导小鼠的免疫应答  

Construction of Neisseria surface protein A gene vaccine of Neisseria gonorrhoeae and evaluation of the immune responses induced by this vaccine in mice model

作　　者：谢良伊[1] 胡四海[1] 唐湘云[2] 杨胜辉[1] 余敏君[1] 韩福郎[1] 

机构地区：[1]南华大学病原生物研究所 [2]湖南环境生物职业技术学院附属医院

出　　处：《中华传染病杂志》2007年第7期392-397,共6页Chinese Journal of Infectious Diseases

基　　金：湖南省自然科学基金(05jj30045);衡阳市科研基金(2005KS01-057)

摘　　要：目的构建淋病奈瑟球菌表面蛋白A(NspA)基因疫苗,并接种小鼠,评价其诱导的体液和细胞免疫应答。方法将NspA基因插入到真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)/NspA,并经PCR、双酶切及测序鉴定。反转录-聚合酶链反应(RT-PCR)和免疫组织化学法分别检测NspA基因转染细胞后mRNA和蛋白的表达。以pcDNA3.1(+)/NspA重组质粒免疫45只雄性BALB/c小鼠,试管凝集法检测免疫小鼠抗体滴度,EIASA检测IFN-γ水平,四甲基偶氮唑蓝(MTT)比色法检测脾淋巴细胞增殖。提取接种部位股四头肌总DNA,PCR检测BALB/c小鼠肌细胞内NspA基因。结果成功构建pcDNA3.1(+)/NspA基因疫苗,能在真核细胞中转录和表达。pcDNA3.1(+)/NspA免疫组的抗体滴度达1:640,pcDNA3.1(+)和PBS免疫组均无特异性抗体检出。空载体pcDNA3.1(+)组IFN-γ为(23.79±11.85)pg/mL,pcDNA3.1(+)/ NspA免疫组为(169.71±30.52)pg/mL(P<0.01);脾淋巴细胞增殖的刺激指数(SI)分别为1.05±0.30和1.94±0.74(P<0.01)。并证实NspA基因可在小鼠肌细胞中稳定存在。结论构建淋病奈瑟球菌NspA基因疫苗,将其免疫BALB/c小鼠可诱导特异性体液和细胞免疫应答,为进一步用于淋病预防奠定了基础。Objective To construct the Neisseria surface protein A （NspA） DNA vaccine of Neisseria gonorrhoeae and evaluate the humoral and cellular immune responses induced by this vaccine in mice model. Methods The recombinant expression vector pcDNA3.1（＋）/NspA was constructed by inserting NspA gene into the eukaryotic expression vector pcDNA3.1 （＋） and confirmed by polymerase chain reaction （PCR）, restriction enzymes Hind Ⅲ, Xba Ⅰ and DNA sequencing. NspA mRNA in transfected RAW264.7 cells and NspA protein expression in transfected COS-7 cells were detected by reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemical stai- ning, respectively. Forty-five male BALB/c mice were immunized with pcDNA3.1 （＋）/NspA recombinant plasmid. The level of serum anti- Neisseria gonorrhoeae antibody of the immunized mice was detected by tube agglutination test, and the level of interferon （IFN）-7 was assayed by enzyme-linked immunosorbent assay （ELISA）. The proliferation of splenocytes was determined by methyl thiazolyl tetrazolium （MTT） colormetry. The NspA gene in BALB/c mice was identified by PCR with the total DNA extracted from quadriceps femoris in immunized sites. Results Restriction enzymes digestion analysis and DNA sequencing results revealed that the pcDNA3. 1 （＋）/NspA had been constructed successfully. NspA gene had been transcripted and expressed in mammalian cells. The peak titer of specific antibody was 1 ： 640 in pcDNA3.1 （＋）/NspA immunized group and there was no specific antibody detected in both pcDNA3.1 （＋） immunized group and PBS group. The IFN-γ level in pcDNA3.1（＋） immunized group was （23.79± 11.85）pg/mL and that in pcDNA3.1（ ＋）/NspA immunized group was（169. 71±30.52）pg/mL （P 〈 0.01）. The stimulation index （SI） of the splenocyte proliferation was 1.05±0.30和 1.94±0.74（P〈0.01） in pcDNA3.1（＋） and pcDNA3.1（＋）/NspA immunized group, respectively, The NspA gene could be detected in

关 键 词：奈瑟球菌 淋病 膜蛋白质类 疫苗 DNA 小鼠 近交BALBC 抗体生成 免疫 细胞 

分 类 号：R392[医药卫生—免疫学]