链重叠PCR构建抗HIV免疫毒素及其表达与鉴定  

Expression and identification of an anti-HIV immunotoxin constructed by overlapping PCR

作　　者：金洪涛[1,2] 金宁一[3] 李臻[1,2] 李昌[3] 付延军[3] 王宏[4] 

机构地区：[1]吉林大学畜牧兽医学院 [2]解放军军事医学科学院全军基因工程重点实验室,长春130062 [3]解放军军事医学科学院全军基因工程重点实验室 [4]暨南大学生命科学技术学院

出　　处：《免疫学杂志》2007年第1期94-96,共3页Immunological Journal

基　　金：全军医药卫生科技攻关项目资助(06G127)

摘　　要：目的提高抗HIV免疫毒素DT-ScFv原核表达的可溶性与生物活性。方法利用链重叠PCR融合的方法,构建了免疫毒素DT-ScFv,二者之间添加了connector序列,免疫毒素基因PCR连接测序以后,将其克隆入pET28a质粒,在BL21(DE3)工程菌中诱导表达,通过免疫印迹鉴定表达的重组蛋白,并通过与表达HIV-1gp120的靶细胞的间接免疫荧光实验,验证免疫毒素的细胞识别结合能力。结果本研究成功地构建了免疫毒素DT-ScFv,经原核表达证明目的蛋白以部分可溶的形式存在于表达菌中,添加的connector序列有助于DT-ScFv在细胞中获得正确折叠,重组免疫毒素能与表达gp120的细胞发生特异性的识别结合。结论利用链重叠PCR融合的方法,添加connector序列,有助于提高免疫毒素表达的可溶性与生物活性。本研究对抗HIV免疫毒素的活性研究与应用研究具有重要的意义。Objective To elevate the soluble expression and bioactivity of anfi-HIV immunotoxin DT-ScFv. Methods The immunotoxin DT-ScFv was constructed by overlapping PCR with connector sequence adding between the two part, and then expressed in pET system. The recombinant protein was confirmed by Western blotting. The cell-recognition ability of DT-ScFv was detected by IFA experiment with the cells expressing HIV-1 gp120. Results The immunotoxin DT-ScFv was constructed and expressed in pET system in a partly soluble way. The added coanector sequence could help the protein fold correctly, thus, the immunotoxin could specific bind with the cells expressing HTV-1 gp120. Conclusion Using of overlapping PCR and addition of connector sequence may help elevate the soluble expression and bioacti-vity of anti-HIV immunotoxin DT-ScFv.

关 键 词：免疫毒素 链重叠PCR 原核表达 

分 类 号：R392[医药卫生—免疫学]