论文部分内容阅读
目的构建抗人卵巢癌单抗ＣＯＣ１８３－Ｂ２单链抗体（ｓｃＦｖ）基因。方法将通过ＰＣＲ方法体外扩增并经测序验证的重链、轻链可变区（ＶＨ、ＶＬ）基因先后重组入原核表达质粒ｐＴＨＡ９０相应的位点上，中间通过一连接肽（Ｇｌｙ４Ｓｅｒ）３基因连接构建成单链抗体基因（ｓｃＦｖ），连接产物转化相应受体菌ＴＯＰ１０，提取质粒，酶切鉴定重组克隆。表达产物经ＥＬＩＳＡ方法测定活性。结果重组克隆经酶切鉴定可见预期大小的片段，表明重组成功。表达产物经ＥＬＩＳＡ检测证实具有与卵巢癌抗原结合的能力。结论本研究成功地构建了ｓｃＦｖ基因，且其表达产物保留了抗体的亲和性和特异性
Objective To construct a single chain anti-human ovarian cancer COC183-B2 scFv gene. Methods The heavy chain and light chain variable region (VH, VL) genes amplified by PCR in vitro and verified by sequencing were recombined into the corresponding sites of the prokaryotic expression plasmid pTHA90 through a Gly4Ser 3 gene Ligated into single-chain antibody gene (scFv), and the ligation product was transformed into the corresponding receptor strain TOP10, the plasmid was extracted, and the recombinant clone was identified by restriction enzyme digestion. The expression product was assayed for activity by ELISA. Results The recombinant clones were identified by restriction enzyme digestion of fragments of the expected size, indicating that the recombination was successful. The expression product was confirmed by ELISA to have the ability of binding with ovarian cancer antigen. Conclusion This study successfully constructed scFv gene, and its expression product retains the affinity and specificity of the antibody