壳聚糖介导甲型副伤寒沙门菌nmpC和pagC DNA疫苗的构建及其免疫效果

Construction and immune effect of chitosan mediated pagC or nmpC DNA vaccine against Salmonella paratyphi A

目的构建壳聚糖(chitosan,CS)介导的甲型副伤寒沙门菌nmpC和pagC的DNA疫苗,并探讨该疫苗的免疫效果。方法按哺乳动物的密码子偏好性对nmpC和pagC的基因序列进行优化,并将优化后的序列克隆至真核表达载体provax中,构建provax-nmpC和provax-pagC DNA疫苗。采用复凝聚法制备载DNA疫苗的CS纳米粒,并对CS/DNA纳米粒的特性进行检测。将provax-nmpC和provax-pagC DNA疫苗及其相应的CS纳米粒体外转染BHK细胞,采用Western blot检测目的蛋白的表达。采用活体电击法分别用不同组分的蛋白免疫小鼠,ELISA法检测血清抗体水平及抗体类型。结果 provax-nmpC和provax-pagC DNA疫苗经双酶切鉴定,构建正确。疫苗可与CS发生有效结合,纳米粒形似球形,粒径均一,包封率均达90%以上,且CS可防止DNA被DNaseⅠ酶降解。provax-nmpC及CS/provax-nmpC均能诱导小鼠产生有效的免疫反应,两组间差异无统计学意义(P>0.05);provax-pagC与CS/provaxpagC均能诱导小鼠产生有效的免疫反应,且provax-pagC组的抗体水平明显高于CS/provax-pagC(P<0.05)。provax-nmpC和provax-pagC组的IgG1及IgG2a抗体水平均高于相应的CS组(P<0.05),且IgG1和IgG2a间差异无统计学意义(P>0.05)。结论 DNA疫苗provax-nmpC和provax-pagC均可诱导有效的免疫反应;载基因CS纳米粒并未增强DNA疫苗的免疫效果;CS未改变DNA疫苗的免疫应答类型,但具有调节Th1和Th2平衡的作用。

Objective To construct a chitosan （CS）-mediated DNA vaccine expressing nmpC orpagC genes of Salmonella paratyphi A and investigate its immune response. Methods The sequences of nmpC and pagC genes were optimized according to the codon-preference of mammals, and cloned into eukaryotie expression vector provax to construct DNA vaccines provax-nmpC and provax-pagC respectively. CS nanoparticles loaded with DNA were prepared by complex coacervation method and analyzed for speciality. BHK cells were transfected in vitro with DNA vaccines provax-nmpC and provax-pagC and the corresponding CS nanoparticles respectively, and determined for expressions of NmpC or PagC by Western blot. Mice were immunized with the recombinant proteins by electric shock, and determined for serum antibody level and types by ELISA. Results Restriction analysis proved that DNA vaccines provax-nmpC and provax-pagC were constructed correctly. The vaccines were bound to CS nanoparticles effectively. The obtained vaccine-loaded nanopartieles were spherical in even size, of which the encapsulation rate was more than 90%. CS prevented DNA from digestion with DNase I . Both provax-nmpC and CS/provax-nmpC induced effective immune response in mice, while the response levels showed no significant difference （P 〉 0. 05 ）. Likely, both provax-pagC and CS/provax-pagC induced effective immune response. However, the antibody level induced by provax-pagC was significantly higher than that by CS/provax-pagC（P 〈 0. 05）. The IgG1 and IgG2a levels induced by provax-nmpC and provax-pagC were higher than those by the corresponding CS-based vaccines （P 〈 0. 05）. However, no significant difference was observed in IgG1 and IgG2a levels. Conclusion Both DNA vaccines provax-nmpC and provax-pagC induced effective immune response in mice. CS nano- particles failed to enhance the effect or alter the type of immune response to DNA vaccine while regulated the balance between Thl and Th2 responses.