幽门螺旋杆菌细胞毒素相关蛋白A真核表达载体的构建与表达

The Construction of the Helicobacter pylori Cytotoxin-Associated Protein A(CagA)Gene Eukaryotic Expression Vector and the Coding Protein Expression

目的构建幽门螺旋杆菌(Helicobacter pylori,Hp)细胞毒素相关蛋白A(cytotoxin-associated protein,CagA)基因表达载体并表达其编码蛋白。方法从胃黏膜活检标本中分离Hp,提取核酸,采用RT-PCR技术扩增CagA基因,将其克隆至pEGFP真核表达载体中,构建Hp CagA基因真核表达载体并转染293-T细胞表达其编码蛋白。结果通过PCR、双酶切、测序鉴定证实CagA基因的真核表达载体构建成功,免疫印迹法证实CagA蛋白的表达。结论成功构建了Hp CagA基因真核表达载体并表达其编码蛋白,为后期功能研究提供了材料和基础。

Objective To construct the Helicobacter pylori cytotoxin associated protein A （CagA） gene eukaryotic expression vector and to express its coding protein. Method Helicobacter pylori from gastric mucosa biopsy specimens was isolated. Helicobacter pylori nucleic acid was extracted and the RT-PCR was used to amplify CagA gene, followed by cloning it into PEGFP eukaryotic expression vector, and transfected it onto 293-T ceils to express the coding protein. Results By the use of PCR,double enzyme digestion,and DNA sequencing, the CagA gene eukaryotic expression vector was constructed successfully and immunoblotting confirmed CagA protein expression. Conclusion We are successful in construction of Helicobacter pylori CagA gene eukaryotic expression vector and in the expression of the encoded protein,thus, providing the foundation for further studies.