中文摘要：

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克隆分别克隆人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同时克隆人真核表达载体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，转化人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HpHpaA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基因克隆人载体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30000和14000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。

英文摘要：

Objective To construct a nucleic acid vaccine expressing H. pylori HpaA and interleukin-2 gene and to identify the immunogenicity of the vaccine proteins in vitro and protection in vivo. Methods The HpaA gene fragment was amplified by polymerase chain reaction（PCR） from the genomic DNA of the standard H. pylori strain 17874. Mouse interlukin（IL）-2 gene was amplified from pCIneoIL-2. The HpaA and IL-2 were cloned into pUCmT vector. After DNA sequences of the amplified HpaA gene and IL-2 were confirmed, both were cloned into the eukaryotic expression vector pIRES through a serial of enzyme digestion and ligation reactions. The recombinant plasmids were screened by PCR and restriction enzyme digestion. Then, recombinant pIRES-HpaA-IL-2 was transfected to COS-7 cells using LipofectamineTM2000. The immunogenicity of HpaA and IL-2 protein was detected by SDSPAGE and Western blot. The recombinant plasmids were transformed to LBS000 and then to final host SL7207. The recombinant strains were passaged repeatedly. The mice were challenged with H. pylori after 4 weeks of inoculation of nucleic acid vaccine. H. pylori infection was detected by rapid urease test. Results The amplified HpaA gene fragment and IL-2 were confirmed by sequence analysis. The eukaryotic expression vector pIRES and the pIRES-HpaA-IL-2 construction were confirmed by PCR and restriction digestion. The expressions of HpaA（30 000） and IL-2 （14 000）protein by pIRES-HpaA-IL- 2 were detected by Western blot. The in vivo study showed that 75.0% and 58.4% of mice vaccinated by HpaA IL- 2 and HpaA, respectively, were protected anaigst H. pylory infection, which was significant different in comparison with PBS control （P 〈 0.0 1 ）. Conclusions A recombinant attenuated nucleic acid vaccine HpaA-AL- 2 encoding both HpaA and mouse IL-2 was successfully constructed and its immunogenicity in vitro and protection in vivo were confirmed. The IL-2 can enhance the protective efficacy in immunized mice.