幽门螺杆菌vacA毒性片段与cagA融合基因的构建与表达  

Construction of fusion gene expressing vacuolating segment of vacA and cagA from Helicobacter pylori

作　　者：林珊珊[1] 杨致邦[1] 刘淼[2] 吴利先[1] 

机构地区：[1]重庆医科大学微生物学教研室,重庆400016 [2]安徽医科大学病原生物学教研室

出　　处：《中国人兽共患病杂志》2004年第12期1032-1035,1044,共5页Chinese Journal of Zoonoses

摘　　要：目的　构建幽门螺杆菌空泡毒素 (VacA)毒性片段和细胞毒素相关蛋白 (CagA)的融合基因 (vlc) ,在原核细胞中表达 ,为Hp双价融合蛋白候选疫苗的研究提供材料。 方法　用GeneSOEing技术将vacA毒性亚单位片段 (v)与cagA保守片段 (c)用疏水性多肽接头 (Gly4Ser) 3 进行拼接 ,构建融合基因vlc,将vlc定向插入原核表达质粒pQE30 ,经DNA测序分析确认后 ,转化E .coliDH5a ,IPTG诱导表达 ,Westernblot分析其抗原性。结果　DNA序列分析表明融合基因的连接顺序、方向正确 ,有一个碱基发生了无义突变。工程菌诱导后可表达相对分子量为 5 8× 10 3 的融合蛋白 ,与预期分子量一致 ,约占菌体总蛋白的 8%。Westernblot显示融合蛋白具有良好的抗原性。结论　融合基因vlc构建成功 ,表达的融合蛋白具有良好的抗原性 ,有望进一步进行免疫保护性机制的研究和Hp疫苗的制备。To constructed the fusion gene of vacuolating segment of vacA and conservative region of cagA and expressed fusion protein in E.coli DH5a.recombinant Gene SOEing was used to constructed the fusion gene of vacuolating segment of vacA gene and conservative region of cagA gene. A hydrophobic polypeptide linker (Gly 4Ser) 3 was used to splice two different gene fragments for construction of fusion gene. And the fusion gene was inserted into prokaryotic expressing vector pQE30.After sequencing,the recombinant plasmid was transformed into E.coli DH5a.The transformant colony was induced with IPTG. Expression of fusion protein was analyzed by SDS PAGE and Western blot. DNA sequence analysis showed that the splicing order and direction were completely correct. There was one base mutation but amino acid radicle which encoded was not changed.When engineered bacteria was induced by IPTG the anticipated Mr 58×10 3 protein band appeared on SDS PAGE gel accounting to 8% of total bactarial proteins. Western blot analysis of the fusion protein confirmed its strong immunogenicity.In conclusion the recombinant plasmid of vacuolating segment of vacA and conservative region of cagA was successfully constructed and expressed. The fusion protein could be used in the further studies of development of Helicobacter pylori vaccine.

关 键 词：幽门螺杆菌 空泡毒素 细胞毒素相关蛋白 融合基因 表达 

分 类 号：R378[医药卫生—病原生物学]