沙眼衣原体主要外膜蛋白_(21-387)的原核表达及其免疫原性

Prokaryotic expression and immunogenicity of major outer membrane protein 21-387 from Chlamydia trachomatis

目的:探讨沙眼衣原体(Ct)E血清型主要外膜蛋白(MOMP21-387)的原核表达及其免疫原性。方法:利用PCR方法扩增Ct E血清型MOMP第21至第387氨基酸的基因序列,克隆至p ET21a(+)原核表达载体构建重组质粒p ET21a(+)/MOMP21-387,并进行原核表达和纯化,经SDS-PAGE和Western blot法分析鉴定后,通过BALB/c小鼠免疫检测MOMP21-387蛋白的免疫原性,即通过ELISA法检测小鼠血清Ig G和生殖道分泌物Ig A抗体反应,乳酸脱氢酶(LDH)法检测其脾细胞的特异性杀伤作用。结果:在原核表达系统成功表达了MOMP21-387融合蛋白,经SDS-PAGE及Western blot法鉴定在相对分子质量(Mr)约44 000处出现特异性条带;并经NiNTA亲和层析的方法获得了纯化的MOMP21-387融合蛋白,免疫BALB/c小鼠可诱导产生特异性血清Ig G抗体和生殖道分泌物Ig A抗体,至第6周达到高峰,MOMP21-387组的Ig G和Ig A抗体较PBS组差异均有统计学意义(P<0.05);LDH检测结果显示,MOMP21-387组小鼠脾细胞对靶细胞的杀伤率,在10:1、20:1和40:1和80:1时均明显高于PBS组,差异有统计学意义(P<0.05)。结论:Ct E型MOMP21-387融合蛋白具有良好的免疫原性,为基于MOMP21-387的Ct的ELISA法检测方法的开发和疫苗研究奠定了基础。

Objective：To explore prokaryotic expression and immunogenicity of major outer membrane protein （MOMP21-387） of Chlamydia trachomatis serotype E. Methods：The gene encoding MOMP21-387 was ampliifed from genome DNA of C. trachomatis E by PCR analysis, and then cloned into pET21a （＋） vector to construct recombinant plasmid pET21a （＋）/MOMP21-387, and was expressed in the prokaryotic expression system. The MOMP21-387 fusion protein was puriifed and identiifed by SDS-PAGE and western blot analysis. The immu-nogenicity was further assessed by immunizing BABL/c mice, the reactivity of speciifc serum IgG in serum and genital tract mucosal IgA were tested by ELISA, and the speciifc cytotoxicity of spleen cells was detected with lactate dehydrogenase （LDH） method. Results：The MOMP21-387 fusion protein was successfully expressed in a prokaryotic expression system, and the speciifc positive bands at the relative molecular mass （Mr） of about 44 000 was conifrmed by SDS-PAGE and western blot analysis;And the puriifed MOMP21-387 fusion protein was ob-tained with the Ni-NTA afifnity chromatography method. The mice can be induced to produce the speciifc serum IgG and reproductive tract mucosal IgA by immunizing with the MOMP21-387 fusion protein, and the value of the speciifc serum IgG and mucosal IgAin immunized groups were signiifcantly higher than that of the PBS control group （P〈0.05）, the antibodys detected by ELISA reached peak at the 6th week post-immunization. The LDH analysis showed that, the killing rate of spleen cells to the target cell in MOMP21-387 protein immunized groups was signiifcantly higher than those in PBS control group, when the effector cells to target ratio reached to 10：1, 20：1, 40：1 and 80：1 （P〈0.05）. Conclusion：The MOMP21-387 fusion protein of Chlamydia trachomatis serovar E＆amp;nbsp;shows good immunogenicity, It may lay the foundation for the Chlamydia trachomatis ELISA detection and vac-cine development based on MOMP21-387.