不可分型流感嗜血杆菌P6重组蛋白的原核表达及其黏膜免疫效应

Prokaryotic expression of recombinant protein P6 of Nontypeable Haemophilus influenzae and analysis of mucosal immunity effect of expressed protein

目的:研究不可分型流感嗜血杆菌重组P6蛋白的免疫原性,以确定重组P6蛋白在不可分型流感嗜血杆菌疫苗研制中的应用价值。方法:扩增P6目的基因,构建重组质粒PGEX-6P2/P6,鉴定后将其转化入E.coli XL1-Blue,IPTG诱导、纯化后经SDS-PAGE、Western blot分析;纯化的重组蛋白经滴鼻方式免疫BALB/c小鼠,ELISA法检测血清IgG,肺灌洗液IgA的水平及脾细胞中IL-4、IL-10、IL-17和IFN-γ的产生水平。CCK-8法检测脾淋巴细胞增殖情况。结果:成功构建了重组表达载体PGEX-6P2/P6,并表达GST-P6融合蛋白。Western blot证明GST-P6蛋白能与菌源P6蛋白免疫小鼠后的抗血清发生特异性反应。动物实验表明重组P6蛋白通过滴鼻途径既能刺激小鼠产生血清IgG,又能诱导肺黏膜产生较高的特异性IgA抗体。重组蛋白+IL-2组小鼠脾淋巴细胞所产生的IL-17和IFN-γ的含量高于对照组(P<0.05)。结论:P6蛋白的原核表达载体PGEX-6P2/P6在大肠杆菌XL1-Blue中大量表达,重组P6蛋白经黏膜免疫可以同时诱导产生体液免疫和细胞免疫。此实验为P6蛋白疫苗的研发提供了理论基础。

Objective：To clone and express outer membrane protein P6 of Nontypeable Haemphoilus influenzae,and detect immunogenicity of P6 recombinant protein,then determine the value of the recombinant P6 protein in NTHi vaccine development.Methods：The P6 gene was amplified with template of NTHi chromosomal DNA by PCR and inserted into a prokaryotic vector PGEX-6P2 to generate PGEX-6P2/P6,which was verified by PCR,enzyme digestion analysis and DNA sequencing.The PGEX-6P2/P6 plasmid was transformed into E.coli XL1-Blue and IPTG was used to induce its expression.SDS-PAGE and Western blot were used to verify the expressed protein.BALB/c mice were intranasally immunized with purified recombinant protein P6.Then indirect ELISA was used to detect the levels of IgG,IgA IL-4,IL-10,IL-17 and IFN-γ.The proliferation of spleen lymphocyte was analyzed by CCK-8 counting.Results：The prokaryotic recombinant plasmid PGEX-6P2/P6 was constructed and P6 protein was successfully expressed under the induction of IPTG.Western blot validated the specific reaction between GST-P6 and anti-sera from P6 immunized mice.P6 protein could stimulate the production of specific IgG and IgA antibodies in lung mucosa.In the mice immunized by recombinant protein plus IL-2,the average concentrations of IL-17 and IFN-γ were significantly （P ＜ 0.05） higher than those in the control mice.Conclusion：The prokaryotic recombinant plasmid PGEX-6P2/P6 was constructed and P6 protein was successfully expressed under the induction of IPTG.Recombinant protein had good immunogenicity and could stimulate humoral and cellular immune responses from mucosal immune.Our research is fundamental for the development of P6 protein vaccine of NTHi.