问号赖型钩体重组质粒rpDJt表达蛋白P68免疫原性的研究

Immunogenecity of Expressed Protein p68 from Recombinant Plasmid rpDJt in L. interrogans serovar lai

为构建一种新型钩体重组亚单位疫苗，采用问号钩体ｒｐＤＪｔ基因的蛋白表达物Ｐ６８免疫动物，对其免疫原性进行研究。结果显示：在体外，Ｐ６８能与其特异性抗血清及赖型钩体死菌苗抗血清呈明显高效价Ａｇ－Ａｂ反应；在体内，Ｐ６８能激起明显的Ｔ淋巴细胞转化，ＴＨ细胞活化，ＩＬ－２，ＩＬ－６活性增加。提示：ｒｐＤＪｔ表达蛋白Ｐ６８能激起机体强有力的Ｔ－Ｂ细胞协同性特异性体液免疫反应，是致病性强毒力钩体重组亚单位疫苗好的候选抗原之一。

There are two types of infection caused by pathogenic microorganisms:intracellular infection and intercellular infection. Infection of pathogenic leptospira is an intercellular infection. The immunological reaction of host to intercellular infection is unique. The potential immunogen of an expressed protein should meet three criteria: it can be degraded (by antigen present cells in the host); it should have antigenic epitope which can be recognized by specific antibodies and have at least one epitope that can be recognized by an MHC Ⅱ protein and T cell receptor. In this study we report the cloning of an L. interrogans protein in plasmid rpDJt and the immunogencity of the expressed protein derivative. A genomic library of L. interrogans serovar lai strain 017 was constructed with the plasmid vector pUC18. Recombinant plasmids, designated pDJH2 and pDJ8 were screened from the bank. EcoRI inserted fragment of 1.9kb recombinant DNA of pDJH2 was ligated into T7 RNA polymerase/promoter vectors (pT7 7). Then they were transformed into E.coli JM109 (De3), one of subclones, designated rpDJt was achieved. SDS PAGE showed that the molecular weights of expression proteins were 68kd and 23kd respectively, designated p68 and p23. Purifying and isolating p68 and p23, we separated them from SDS Polyacrylamide gels by using Side Strip method. After fragmenting and electroeluting, p68 and p23 were injected into guinea pigs and rabbits. An extremely strong immune response to p68 was obtained since an anti p68 antibody response could be detected to a dilution 1∶524288 (guinea pigs) and 1∶262144(rabbits) by ELISA while anti P23 antibody being 1∶1024 (the same to guinea pigs and rabbits). The results of improved MTT and conA 3HTdR transformation methods showed the activities and proliferation of Th cells were increased in guinea pigs after p68 immunization (IL 6, 83.25IU/ml; IL 2, 28.75 IU/ml; RPI, 2.04, SI, 65.62%) Thlymphocyte existed in two subclasses, the Th1 and Th2 cells. A major role of Th2 cells is to “help” B cells differentiate, replicate, and secrete antibody. The properties of these interactions explain why p68 makes good antigen and p23 does not. The antigens responsible for eliciting the production of protective antibodies are not known; however, several outer membrane proteins on L. interrogans are candidates for vaccine. Our results suggest that expresion protein p68 from recombinants (rpDJt) may be a candidate for gene engineered subunit vaccine for Leptospirosis.