免疫途径和佐剂对NTHiP6蛋白疫苗免疫效果的影响

The immunological effects of the inoculation route and type of adjuvant on a vaccine derived from the P6 protein of nontypeable Haemophilus influenzae

目的观察不同免疫途径和佐剂对NTHiP6蛋白疫苗免疫效果的影响。方法将原核表达质粒PGEX-6P2/P6转入E.coli XL1-Blue,IPTG诱导P6蛋白的表达并进行纯化。以不同免疫途径(皮下,腹腔,滴鼻)用P6蛋白免疫小鼠,每组12只。然后另取小鼠分为PBS组和不同佐剂组(MDC、IL-2、弗氏佐剂),每组15只,采用皮下注射途径免疫。共免疫3次,间隔2周,用ELISA检测小鼠血清特异性IgG抗体,用CCK-8法检测脾淋巴细胞增殖活性,用ELISA检测IL-4和IFN-γ水平。结果在大肠埃希菌中成功表达P6蛋白。3种免疫途径相比,皮下途径免疫诱导的IgG抗体滴度为1 279.97±3.87,腹腔免疫组为427.16±3.08,滴鼻免疫组为253.98±3.30,差异有统计学意义(F=5.43,P<0.05)。采用皮下免疫途径时,MDC组诱导的IgG抗体滴度、IL-4水平分别为7 421.02±3.30和79.64±2.75,与其他组比较差异有统计学意义(P<0.05)。IFN-γ水平和脾淋巴细胞增殖指数与IL-2组相比差异无统计学意义(P>0.05)。结论使用MDC佐剂并采用皮下途径注射可使NTHiP6蛋白疫苗获得较好的免疫效果。

Objective To explore the immunological effects of the route of inoculation and type of adjuvant on a vaccine derived from the P6 protein of nontypeable Haemophilus influenzae. Methods The recombinant plasmid PGEX-6P2-P6 expressing NTHiP6was transformed into E.coli XL1-Blue and expression of the P6 protein was induced with IPTG.The fusion P6 protein was then purified.First,BALB/c mice（12mice per group）were inoculated via different routes（intraperitoneally,intranasally,and subcutaneously）.Second,the protein was combined with various adjuvants（Freund’s adjuvant,IL-2,and MDC）and administered（15mice per group）.Mice were immunized 3times with a two-week interval.The titer of serum IgG antibodies was detected using ELISA.In addition,splenocytes were isolated and cultured to monitor cell proliferation with a CCK-8kit and levels of IL-4and IFN-γwere assayed using ELISA. Results The P6 fusion protein was successfully expressed and purified from E.coli XL1-Blue cells.The IgG antibody titer induced by subcutaneous immunization was 1,279.97±3.87,that inducted by intraperitoneal immunization was 427.16±3.08,and that induced by intranasal immunization was 253.98±3.30.Differences in antibody titers were statistically significant（F=5.43,P〈0.05）.When inoculated subcutaneously,the MDC group had a high IgG antibody titer（7 421.02±3.30）as well as a high level of IL-4（79.64±2.75）,and these measurements differed significantly from those in other treatment groups（P〈0.05）.However,there were no differences between the MDC group and the IL-2group in terms of IFN-γlevels and the specific proliferation index（P〉0.05）. Conclusion Subcutaneous injection of the P6 protein and MDC may induce an improved immune response.