摘要
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。
Objective To construct single chain Fv (scFv) gene of anti-human ovarian carcinoma monoclonal antibody (McAb) COC183-B 2. Methods The heavy and light chain variable region genes of the antibody COC183-B 2 were inserted into two corresponding sites of expression vector pTHA90,and a scFv gene was constructed with a short peptide (Gly 4Ser) 3 gene. The recombinants were determined by digesting with Xho Ⅰ/Spe Ⅰ,Xba Ⅰ/EcoR Ⅰ and Xho Ⅰ/EcoR Ⅰ,and then were introduced into E.coli TOP 10 .The antigen binding activity of expressed product was detected with ELISA. Results The recombinants were determined by digesting with endonucleases and expected bands were identified. The value of expressed scFv was 3 times more than negative control by ELISA. Conclusion The scFv gene of COC183-B 2 was successfully constructed and expressed, and the expressed scFv fragment could interact specifically with ovarian carcinoma associated antigen.
出处
《徐州医学院学报》
CAS
1998年第6期446-448,共3页
Acta Academiae Medicinae Xuzhou
基金
国家自然科学基金
关键词
卵巢肿瘤
单链抗体
载体构建
基因表达
Ovarian carcinoma single-chain Fv Construction Expression
