摘要
构建一种可实现外源基因高效的、适用于中华仓鼠卵巢细胞(CHO)细胞的质粒表达系统,并成功实现了恶性疟原虫膜蛋白pfs25基因在CHO细胞中的表达。新构建的真核表达载体是在pBudCE4.1质粒载体上改造获得的,被命名为pCMV-WD,包含了人工合成的、完整的弱化SV40启动子与二氢叶酸还原酶(DHFR)基因组成的顺式表达元件和全新设计的多克隆位点,该载体属于CHO细胞通用载体,理论上可以实现任何外源基因在CHO细胞中的表达。基于该载体,采用定向克隆策略插入了恶性疟原虫膜蛋白pfs25基因,构建的重组表达质粒为pfs25/pCMV-WD。经zeocin抗生素和撤除H、T(次黄嘌呤、胸腺嘧啶脱氧核苷)双筛选,获得了DHFR+基因阳性的细胞克隆。对表达量最高的克隆株进行亚克隆,并经MTX加压筛选以获得高表达pfs25蛋白的CHO细胞株。ELISA和Western Blot结果表明,重组pfs25蛋白具有良好的免疫学反应性,且表达量为0.1 mg/mL。该研究首次获得了可分泌重组pfs25蛋白的CHO细胞株。
Malaria is the mosquito-borne lethal infectious disease. The pfs25 protein was proved to be the antigen for transmissionblocking vaccine. The aim of our study is to construct eukaryotic expressive vector for expression of Plasmodiumfalciparum transmission-blocking vaccine antigen pfs25 in CHO cell. In this study,the novel vector, in which the expression cassette was integrated with weaken SV40 promoter and dihydrofolate reductase （dhfr） ,was constructed and pfs25 gene was cloned into the new vector. The novel vector was designed and accomplished based on the pBudCFA. 1 vector and named as pCMV-WD. It included the artificially synthetica weaken SV40 promoter, dhfr expression cassette and newly designed multiclone sites（MCS）. The vector was applied especially on the CHO cell. The pfs25 gene was inserted into the pCMV-WD vector and recombinant plasmid pfs25/pCMV-WD was constructed, and then transformed into the host CHO-DHFR- cell by electroporation. Positive CHO cells expressed pfi25 gene were obtained by selection of deleting hypoxomthine, thymioline and adding zeocin antibiotics in medium. The recombinant pfs25 protein was identified by ELISA and Western Blot. The positive reaction was found in the Western Blot assay. Seven positive CHO cell lines expressed pfs25 gene were obtained, and the 0. 1 mg/mL pfs25 was detected in the culture medium of 5A4 CHO cell line. The pfs25 gene was expressed successfully in the CHO cell by the eukaryotic expressive vector construction.
出处
《药物生物技术》
CAS
2013年第1期12-16,共5页
Pharmaceutical Biotechnology
