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目的探讨载脂蛋白B单链抗体基因的克隆与表达。方法利用本室制备的抗人载脂蛋白B单克隆抗体杂交瘤细胞株,通过提取RNA及逆转录PCR技术使其形成的重、轻链片段与质粒Puc19载体连接,形成克隆,并对其进行鉴定分析。结果抗体重、轻链可变区扩增拼接后的重、轻链各约为360bp,基因全长约725bp将拼接产物插入Puc109质粒中转化Jm109大肠杆菌,得到数个克隆,经酶切分析约含725bp片段,与拼接结果基本相同,经紫外分光光度计测定DNA浓度,特异性好,与其他杂蛋白及载脂蛋白没有交叉反应。结论抗人载脂蛋白B单链抗体基因的克隆和表达较为成功。
Objective To investigate the cloning and expression of single chain antibody of apolipoprotein B Methods The monoclonal antibody against human apolipoprotein B (McAb) hybridoma cell line was prepared and its heavy and light chain fragments were ligated with the plasmid Puc19 vector by RNA extraction and reverse transcription PCR to form clones. Identification analysis. Results The heavy and light chains of antibody heavy and light chain were about 360 bp in length and about 725 bp in length after splicing. The spliced product was inserted into Puc109 plasmid and transformed into E. coli Jm109. Several clones were obtained and analyzed by restriction analysis Containing 725bp fragment, which is basically the same as the splicing result. The DNA concentration was determined by UV spectrophotometer and the specificity was good. It did not cross-react with other hybrid proteins and apolipoproteins. Conclusion Cloning and expression of anti-human apolipoprotein B scFv gene was successful.