绵羊CTLA-4胞外区对细粒棘球蚴EG95抗原的免疫佐剂作用

Immunoadjuvant effect of extracellular domain of shee CTLA-4 on EG95 antigen of Echinococus granulosus

目的探索绵羊细胞毒性T细胞相关抗原-4(cytotoxic Tlymphocyte-associated antigen4,CTLA-4)胞外区靶向提呈羊细粒棘球蚴(Echinococcus granulosus,Eg)EG95抗原的可行性。方法用RT-PCR扩增CTLA-4胞外区编码序列,插入原核表达载体pET-30a获得融合表达载体pET-IgV;根据蛋白质二级结构分析结果 ,用PCR扩增EG95亲水区编码序列,将串联的3拷贝EG95编码序列分别插入表达载体pGEX-6P-1和pET-IgV,诱导重组大肠杆菌表达并纯化重组蛋白;用相同剂量的GST-3EG95、IgV-3EG95包涵体或可溶性蛋白免疫小鼠,比较EG95抗体的应答水平。结果用RT-PCR扩增得绵羊CTLA-4胞外编码区,IPTG诱导的重组大肠杆菌表达预期的约63kDa GST-3EG95和60kDa IgV-3EG95融合蛋白,两者均能被EG95抗血清识别;经两次免疫后,IgV-3EG95免疫组的EG95抗体水平显著高于GST-3EG95免疫组,可溶性蛋白的免疫效果优于包涵体。结论绵羊CTLA-4胞外区可作为分子佐剂促进细粒棘球蚴EG95抗原的抗体应答水平。

To investigate adjuvant effect of the extracellular domain of shee T lymhocyte-associated antigen 4（CTLA-4） on the EG95 antigen of granulosus,the cDNA for extracellular domain of CTLA-4 was amlified from blood lymhocytes of shee by RT-CR.Then the 0.4-kb cDNA was inserted in rokaryotic exression vector ET-30a to obtain fusion exression vector ET-IgV.According to the secondary structure rediction of EG95 rotein,the hydrohilic amino acid region with high antigenic index was amlified by CR,and 3 coies of the CR roduct were inserted into tandem into rokaryotic exression vector GEX-6-1 or ET-IgV.After ITG induction,the recombinant E.coli exressed an exected fusion rotein GST-3EG95 or IgV-3EG95 based on SDS-AGE analysis.Western blotting results showed that the exressed roteins were recognized by anti-EG95 serum.Fifteen mice were immunized twice with 50μg of fusion rotein GST-3EG95 or IgV-3EG95 in inclusion body or soluble form.Indirect ELISA showed that the anti-EG95 antibody was detectable from week 2 in IgV-EG95-immunized mice with a highest titer of 1∶10 000（soluble rotein immunized grou） or 1∶6 400（inclusion body-immunized grou） at week 5 after rimary immunization.In GST-3EG95-immunized mice,the secific antibody was detectable from week 3 with a highest titer of 1∶3 200 without significant difference between the inclusion body and soluble rotein-immunized grous.These data demonstrate that the extracellular domain of shee CTLA-4 has strong immunoadjuvant effect on EG95 antigen of Echinococus granulosus.