摘要
目的构建二硫键稳定单链抗体(B3dsscFv)融合PE38原核表达载体,实现其高效表达,并对初步纯化后的复性产物的稳定性、对肿瘤细胞的结合和杀伤活性进行测定。方法重叠PCR连接B3抗体的VH和VL片段,并将PCR产物克隆至pET22b表达载体(B3dsscFv-pET22b)。测序正确的PE38基因片段经酶切连接至B3dsscFv-pET22b,构建B3dsscFv-PE38-pET22b表达载体。IPTG诱导转化菌,将表达的包涵体进行变性、复性后用阳离子柱Q-Sepharose进行了初步纯化,ELISA测定此融合蛋白中单链抗体的结合活性及其在37℃的稳定性,并采用MTT法检测其对B3抗原表面阳性细胞HT-29的杀伤作用。结果双酶切鉴定表明成功地构建了B3dsscFv-PE38-pET表达载体,表达产物以包涵体形式存在,占总蛋白量的45%左右。复性后的B3dsscFv-PE38保持单链抗体的结合活性,并且对结肠癌HT-29细胞具有一定的杀伤作用,最大杀伤率可达96%。该蛋白在37℃孵育16h后,仍能保持大部分活性。结论所获B3dsscFv-PE38免疫毒素具备导向和杀伤肿瘤细胞的双重功能和良好的稳定性,为其研制有效的肿瘤免疫治疗靶向药物提供一定的基础。
AIM： To construct the expression vector of a recombinant toxin composed of a disulfide stable singlechain antibody from mAb B3 and PE38 and examine the binding ability and cytotoxicity of the purified renatured products against the B3 positive carcinoma cells. METHODS： The VH and VL fragments of the mAb B3 were ligated by overlaping PCR and the resulting product was cloned to the pET22b expression vector. The PE38 fragment was inserted into the B3dsscFv-pET22b expression vector which was digested by EcoR Ⅰ and Hind Ⅲ. The identified expression plasmid was tansformed into E. coli BL21 （DE3） followed by IPTG induction. The inclusion body was purified through QSepharose anion exchange column after denaturing and refolding. The binding and cytotoxic ability of the purified prod- ucts were examined by cell-ELISA and Non-Radioactive Cell Proliferation Assay seperately. The stability assay was performed by incubating the protein sample at 37℃. RESULTS： The expression vector B3dsscFv-PE38-pET was constructed successfully and the expression product existed mainly in the form of inclusion body, accounting for 45% of the totle protein. The refolding product remained the binding ability of the single-chain antibody and exerted cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37℃, CONCLUSION： This genetically engineered B3dsscFv-PE38 fusion protein was stable and bifunctional, tumor targeting and tumor cell killing, supporting that it would be a promising candidate for tumor targeted immunotherapy,
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第1期74-77,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30171091
30271478)
北京市自然科学基金资助项目(No.7022031)
