论文部分内容阅读
利用引物PCR和重叠延伸PCR法,将抗肝癌人源化抗体hscFv25重链可变区和轻链可变区各定点突变出一个半胱氨酸,将hTNFα改造成高抗肿瘤活性的突变体,分别原核表达重链可变区突变体、轻链可变区突变体-突变体hTNFα融合基因,然后将两种表达的产物混合进行复性,目的获得具有高稳定性和高抗肿瘤活性的抗肝癌靶向细胞因子。结果重链可变区突变体和轻链可变区突变体-突变体hTNFα融合基因在大肠杆菌中均获得高效表达表达产物以包涵体形式存在,混合复性结果未获得活性产物。
Using primer PCR and overlap extension PCR, a cysteine was mutated at each site of hscFv25 heavy chain variable region and light chain variable region of humanized HCC antibody, and the hTNFα gene was transformed into a mutant with high anti-tumor activity. The mutants of the heavy chain variable region and the light chain variable region mutant-mutant hTNFα were respectively prokaryoticly expressed, then the two expressed products were mixed for renaturation, and the purpose was to obtain the anti-tumor activity with high stability and anti-tumor activity Liver cancer targets cytokines. Results The recombinant fusion protein of heavy chain variable region and light chain variable region mutant - mutant hTNFα was highly expressed in E.coli and existed as inclusion body. The mixed refolding results showed no active product.