以白细胞介素-2为免疫佐剂的幽门螺杆菌尿素酶B亚单位核酸疫苗的构建及其免疫保护作用

背景：细胞因子具有免疫佐剂效应，但将其用作幽门螺杆菌（H．pylori）核酸疫苗佐剂的研究报道尚少。目的：构建同时含H．pylori尿素酶B亚单位（ureB）基因和小鼠白细胞介素-2（IL-2）基因的重组活减毒鼠伤寒沙门菌核酸疫苗，体外鉴定其表达蛋白的免疫原性，体内检测其对H．priori感染的免疫保护作用。方法：以聚合酶链反应（PCR）扩增H．priori ureB基因和小鼠IL-2基因，分别插入pUCmT载体，测序，通过一系列酶切、连接反应分别克隆人真核表达载体pIRES，酶切、PCR鉴定；重组质粒pIRES-ureB和pIRES-ureB-IL-2分别转化减毒鼠伤寒沙门菌LB5000，抽提质粒，进一步转化终宿主菌SL7207，反复传代培养。以Lipofectamine^TM2000将重组质粒分别体外转染COS-7细胞，蛋白质印迹法检测表达蛋白的免疫原性。以疫苗菌经口接种小鼠，4周后予H．pylori攻击，攻击后4周鉴定H．pylori感染状况。结果：测序结果显示扩增出的ureB和IL-2基因序列与GenBank中的H．pylori ureB和小鼠IL-2序列一致，酶切、PCR鉴定证实ureB和IL-2基因已克隆人pIRES载体，并成功构建了稳定的含H．pylori ureB和小鼠IL-2基因的重组活减毒鼠伤寒沙门菌核酸疫苗。蛋白质印迹法显示，pIRES-ureB-IL-2转染的COS-7细胞表达特异性UreB和IL-2蛋白。体内实验显示，疫苗接种组小鼠的免疫保护率显著高于PBS对照组（P〈0．01），其中ureB-IL-2疫苗组又显著高于ureB疫苗组（87．5％对62．5％，P〈0．05）。结论：成功构建了编码H．pylori ureB和免疫佐剂IL-2基因的重组活减毒鼠伤寒沙门菌核酸疫苗，体外实验证实其可表达具有免疫原性的抗原蛋白和佐剂蛋白，体内实验证实其对小鼠H．pylori感染具有免疫保护性，免疫佐剂IL-2可提高核酸疫苗的免疫保护率。

Construction of Helicobacter pylori Urease B Subunit Nucleic Acid Vaccine with Interleukin-2 as Immunologic Adjuvant for Immunoprotection of Helicobacter pylori Infection

Background:Cytokines can be used as immunologic adjuvant,however it is seldom reported in the study of Helicobacter pylori(H.pylori) nucleic acid vaccine.Aims:To construct a recombinant live attenuated Salmonella typhimurium(S.typhimurium) nucleic acid vaccine encoding H.pylori urease B subunit(ureB) gene and mouse interleukin-2(IL-2) gene and identify the immunogenicity of the proteins expressed in vitro and the prophylactic protection for H.pylori infection in vivo.Methods:ureB gene fragment of H.pylori and mouse IL-2 gene were amplified by polymerase chain reaction(PCR) and then cloned into pUCmT vector,respectively.After sequence analysis,both were cloned into eukaryotic expression vector pIRES through a series of enzyme digestion and ligation reactions.The recombinant plasmids pIRES-ureB and pIRES-ureB-IL-2 were identified by restriction enzyme digestion and PCR,and then transformed the attenuated S.typhimurium LB5000.The plasmids extracted from LB5000 were used to transform the final host SL7207 and the vaccine strains were passaged in LB medium repeatedly.Then,the recombinant plasmids were transfected into COS-7 cells in vitro using LipofectamineTM 2000,the immunogenicity of UreB and IL-2 proteins expressed was detected by Western blot.Four weeks after oral inoculation with vaccine strains,the mice were challenged with H.pylori,and the positivity rate of H.pylori infection was detected 4 weeks after challenge.Results:Sequence analysis showed that the ureB gene fragment and IL-2 gene amplified were consistent with the sequence of H.pylori ureB and mouse IL-2 reported by GenBank,respectively.With restriction enzyme digestion and PCR,it was confirmed that ureB gene and IL-2 gene were cloned into vector pIRES and a stable recombinant live attenuated S.typhimurium nucleic acid vaccine encoding H.pylori ureB gene and mouse IL-2 gene was successfully constructed.Specific strips of UreB and IL-2 proteins expressed by pIRES-ureB-IL-2 transfected COS-7 cells were detected through Western blot.The study in vivo showed that the protection rate of vaccinated groups was significantly higher than that of the PBS control(P<0.01),and that of ureB-IL-2 group was higher than that of ureB group(87.5% vs.62.5%,P<0.05).Conclusions:A recombinant live attenuated S.typhimurium nucleic acid vaccine encoding both H.pylori ureB gene and immunologic adjuvant IL-2 gene is constructed successfully and its immunogenicity in vitro and prophylactic protection in vivo are confirmed in the present study.IL-2 as an immunologic adjuvant may enhance the protective efficacy in the immunized mice.