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目的构建含幽门螺杆菌(Hp)粘附素(HpaA)基因和白细胞介素(IL)-2的核酸疫苗,体外转染COS-7细胞,鉴定其表达蛋白的免疫原性和免疫保护作用。方法应用聚合酶链反应(PCR)技术从Hp标准菌株CCUG17874基因组DNA扩增HpaA基因;从重组质粒pCIneo-IL-2扩增小鼠IL-2基因,并通过TA克隆分别克隆入pUCmT载体。检测HpaA及IL-2的核苷酸序列,酶切、连接反应将HpaA和IL-2同时克隆入真核表达载体pIRES,再经PCR法和酶切反应进行鉴定;通过脂质体法将重组载体pIRES-HpaA-IL-2转染COS-7细胞,SDS-PAGE及Western印迹法检测表达蛋白的免疫原性。重组载体转化减毒鼠伤寒沙门菌LB5000,抽提质粒,转化入SL7207,反复传代,鉴定重组核酸疫苗菌的稳定性。以该疫苗菌经口接种小鼠,4周后再用Hp攻击,鉴定感染状况。结果测序结果证实扩增的HpaA基因与Hp HpaA序列一致,IL-2序列和小鼠IL-2序列一致。PCR和酶切鉴定结果证实,HpaA和IL-2基因克隆入载体pIRES,成功构建含HpaA和IL-2基因的核酸疫苗质粒pIRES-HpaA-IL-2,Western印迹法检测到相对分子质量分别为30 000和14 000的HpaA和IL-2蛋白条带。小鼠体内实验显示HpaA-IL-2及HpaA组分别有75.0%、58.4%获免疫保护,与PBS组差异有统计学意义(P<0.01)。结论成功构建了HpaA和IL-2的Hp减毒沙门核酸疫苗菌,其免疫原性和保护性均得到证实,免疫佐剂IL-2可提高免疫保护率。
Objective To construct a nucleic acid vaccine containing HpaA gene and interleukin (IL) -2 and transfect COS-7 cells in vitro to identify the immunogenicity and immunoprecipitation of the expressed protein. Methods The HpaA gene was amplified from genomic DNA of Hp standard strain CCUG178 by polymerase chain reaction (PCR). The mouse IL-2 gene was amplified from recombinant plasmid pCIneo-IL-2 and cloned into pUCmT vector by TA cloning. The nucleotide sequences of HpaA and IL-2 were detected. The HpaA and IL-2 were cloned into the eukaryotic expression vector pIRES at the same time by enzyme digestion and ligation reaction, and then identified by PCR and restriction enzyme digestion. The recombinant The vector pIRES-HpaA-IL-2 was transfected into COS-7 cells, and the immunogenicity of the expressed protein was detected by SDS-PAGE and Western blot. Recombinant vector was transformed into attenuated Salmonella typhimurium LB5000, plasmid was extracted, transformed into SL7207, repeatedly passaged to identify the stability of recombinant nucleic acid vaccine. Mice were vaccinated with the vaccine and challenged with Hp 4 weeks later to identify the infection. Results The sequencing results confirmed that the amplified HpaA gene was consistent with the Hp HpaA sequence. The IL-2 sequence was consistent with the mouse IL-2 sequence. PCR and restriction enzyme digestion confirmed that the HpaA and IL-2 genes were cloned into vector pIRES, and the recombinant plasmid pIRES-HpaA-IL-2 containing HpaA and IL-2 gene was successfully constructed. The relative molecular mass of HpaA and IL- 30 000 and 14 000 HpaA and IL-2 protein bands. In vivo experiments in mice showed that 75.0% and 58.4% of HpaA-IL-2 and HpaA groups were immunoprecipitated, respectively, with statistical significance (P <0.01). Conclusions Hp attenuated Salmonella DNA vaccine strains with HpaA and IL-2 were successfully constructed. The immunogenicity and protection of HpaA and IL-2 were all confirmed. Immune adjuvant IL-2 could improve the immunoprotection rate.