完整人抗-D抗体分子表达载体的构建

Construction of the Recombinant Expression Plasmid for the Expression of the Whole Anti-D Immunoglobulin Molecules

目的构建含完整人抗-D抗体重、轻链基因的表达载体,为体外表达完整的抗-D抗体分子打下基础。方法从1株分泌IgM抗-D的细胞株提取总RNA,以随机引物逆转录成cDNA,用所设计的抗体前导区引物进行扩增,克隆、测序后将抗-D抗体重、轻链可变区基因分别和带有人IgG1恒定区和к轻链恒定区的表达载体连接,转化大肠杆菌后提取质粒DNA,用PCR和酶切进行鉴定。结果序列分析发现所扩增的基因符合人Ig的特征,除引导序列外,其余序列和我们以前所发表的Fab段序列一致,PCR和酶切鉴定抗体重、轻可变区基因克隆成功。结论成功地扩增了带引导序列的抗-D抗体可变区基因,并构建了完整人抗-D抗体分子表达载体。

Objective To construct the recombinant expression plasmid for the preduction of recombinant anti- （Rhesus D） antibody in vitro. Methods The total RNA was extracted from an Epstein-Barr virus transformed cell line secreting monoclonal anti-（Rhesus D） antibody. The leader region primers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-（Rhesus D） antibody（IgMκ） variable region genes were amplified. After cloning and subsequent sequence analysis, the heavy chain and light chain variable region were cloned into pAH4604 and pAC,4622, respectively. The recombinants were analyzed by PCR and restriction site analysis. Results DNA fragments of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primers, respectively. Sequence analysis indicated that the deduced amino acid sequences were the same as the amino acid present in the human Ig variable region and our previously published sequences except the leader sequences. The heavy chain and light chain variable region were cloned into the expression vectors successfully. Conclusion The variable regions of heavy and light chain of anti-D were successfully amplh＇ied using designed leader region primers. The expression plasmids for producing the whole immunoglobulin molecules of anti-D were constructed.