结核分枝杆菌Ag85B-Ag85A双抗原融合真核表达质粒的构建及表达

Construction of the fused eukaryotic expression vector of Mycobacterium tuberculosis Ag85B and Ag85A antigens and its expression

目的 :构建结核分枝杆菌分泌蛋白Ag85B Ag85A融合基因及其双价抗原真核表达载体 .方法 :采用 geneSOE ing法将Ag85B和Ag85A编码基因用疏水甘氨酸接头(Gly4Ser) 3 行PCR扩增融合 ,经限制性内切酶消化后克隆入pcDNA3.1 (+)中构建真核表达质粒 pCDAg85B A ,酶切、DNA测序鉴定 ,用脂质体法将 pCDAg85B A转染COS 7细胞 ,采用RT PCR ,ELISA方法检测其表达 .结果 :Ag85B Ag85A融合基因定向克隆入pcDNA3.1 (+) ,双向测序表明碱基无突变 ,Ag85B Ag85A融合基因在真核细胞中能表达 .结论 :成功构建结核分枝杆菌分泌蛋白Ag85B Ag85A融合基因及其双价抗原真核表达载体 。

AIM: To construct eukaryotic expression vector of fused gene encoding Mycobacterium tuberculosis Ag85B and Ag85A. METHODS: Fused gene encoding Ag85B and Ag85A of tubercle bacilli was linked with the(Gly 4Ser) 3 linker by gene SOEing. The fused gene was inserted into pcDNA3.1(+). The recombinant plasmid pCDAg85B A was transfected into COS 7 cells with liposome. The expression was detected with RT PCR and ELISA. RESULTS : The fused gene encoding Ag85 and Ag85A was cloned into pcDNA3.1(+) correctly, and no mutation was observed, and its expression was determined in COS 7 cells. CONCLUSION: Eukaryotic recombinant plasmids encoding fused gene of Ag85B and Ag85A antigens were constructed successfully. The results established the basis for further study of the DNA vaccine against tuberculosis.