黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位优化的淋病奈瑟菌孔洞蛋白B核酸疫苗的构建及其诱导小鼠的免疫应答

Construction of DNA vaccine with neisseria gonorrhoeae porin B fused with B subunit of Echerichia coliheat-labile enterotoxin and study on its immune responses in mouse

目的分析黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位（LTB）辅佐的淋病奈瑟菌孔洞蛋白B（PorB）核酸疫苗诱导小鼠的免疫应答水平。方法PCR法扩增目的基因porB、ltB、ltB-porB，分别构建3种相应的pcDNA3．1（一）真核重组表达载体，经PCR、双酶切及基因测序鉴定后转染Hela细胞，用细胞免疫荧光法鉴定质粒的蛋白表达。将核酸疫苗经鼻饲免疫雌性BALB／c小鼠，检测体液免疫及细胞免疫应答水平，同时用免疫组织化学法检测porB、ltB、ltB-porB基因在小鼠鼻黏膜内的表达。组间均数比较采用方差分析。结果构建的真核重组质粒均能在Hela细胞内及小鼠鼻黏膜组织内表达。核酸疫苗免疫组小鼠的生殖道灌洗液PorB特异性sIgA及血清porB特异性IgG水平明显高于对照组（P〈0.01；P〈0.05），且ltB-porB融合基因组特异性sIgA明显高于porB组（P〈0.05）；pcDNA3.1（一）／porB组小鼠脾淋巴细胞培养上清液中IFN-γ、IL-4和脾淋巴细胞刺激指数（SI）分别为（170.04±23.89）pg／mL、（114.68±14.27）pg／mL和1.68士0.19，pcDNA3.1（一）／ltB-porB组分别为（161.42±27.50）pg／mL、（124.16±19.04）pg／mL和1.73±0.28，均高于对照组pcDNA3.1（一）和PBS，差异均有统计学意义（P〈O.01；P〈0.05），高于对照组pcDNA3.1（一）／ttB，差异均有统计学意义（均P〈0.05），但两核酸疫苗免疫组间差异无统计学意义（均P〉0．05）。结论所构建的核酸疫苗分别在Hela细胞及小鼠鼻黏膜组织内获得了表达，经黏膜途径免疫能诱导小鼠产生较高水平的特异性体液免疫和细胞免疫应答，尤其是黏膜免疫应答；证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道黏膜免疫。

Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B （PorB） fused with B subunit of Escherichia coli heat-labile enterotoxin B （LTB） in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction （PCR） and cloned into eukaryotic vector pcDNA3.1（--）. The recombinants were identified by PCR, enzyme digestion and DNA sequencing. The vectors were transfected into Hela cells, and expressed proteins were checked bycytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay （ELISA） and methyl thiazolyl tetrazolium （MTT） colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls （P〈0.01; P〈0.05）. Moreover, the slgA level in pcDNA3, l（--）/ltPrporB group was higher than that in pcDNA3. 1 （-）/porB group （P= 0. 002）. The levels of interferomgamma （IFN-7） and interleukin-4 （IL-4） in the supernatants and stimulation index （SI） of spleen lymphocyte culture in pcDNA3, l（--）/porB group were （170.04±23.89） pg/mL, （114.68±14.27） pg/mL and 1.68±0.19, respectively; and those in pcDNA3, l（--）/ltB-porB group were （161.42±27.50） pg/ mL, （124.16±4_19.04） pg/mL and 1.73±0.28, respectively~ which were both higher than those in pcDNA3.1（--）/ phosphate buffered saliae （PBS） group （P〈0.01; P〈0.05） and pcDNA3. 1（ ）/ ltB group （all P〈0.05）, while there was no significant difference between pcDNA3.1（ ）/ltB-porB group and pcDNA3, l（--）/porB group （0.998, 0. 696, 0. 994;all P〉0.05）. Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 （ -- ）/porB and pcDNA3. 1 （ -- ）/ltB- porB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.