摘要
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.
出处
《第四军医大学学报》
北大核心
2003年第21期1973-1975,共3页
Journal of the Fourth Military Medical University
基金
重庆市卫生局重点项目 (0 0 1 0 0 6)