论文部分内容阅读
应用氯化铯-超离心法从抗人纤维蛋白单克隆抗体SZ-63杂交瘤细胞抽取总RNA,经Oligo(dT)纤维素柱层析分离出mRNA。以mRNA为模板,分别以3′VH和3′Cκ为逆转录引物,在M-MLV逆转录酶作用下,合成重链可变区和轻链可变区第一条链cDNA。然后,采用PCR技术,扩增出了SZ-63重链和轻链可变区基因。将其分别克隆至pUCl8载体中,筛选阳性克隆。经用~(32)P标记双脱氧末端终止法核苷酸序列分析,显示SZ-63重链可变区长为354bp,编码118个氨基酸,轻链可变区长为321bp,编码107个氨基酸。应用基因重组技术将SZ-63可变区基因片段与人免疫球蛋白γ1重链CHl和κ轻链恒区基因进行拼接,构建噬菌体质粒,并在大肠杆菌中表达。表达的嵌合抗体Fab片段为可溶性,经ELISA及Western blot检测,证实表达的嵌合抗体能特异地与人纤维蛋白呈结合反应,表达量约为225μg/L。
Total RNA was extracted from anti-human fibrin monoclonal antibody SZ-63 hybridoma cells by cesium chloride-ultracentrifugation and mRNA was isolated by Oligo (dT) cellulose column chromatography. Using mRNA as a template and 3’VH and 3’Cκ as reverse transcription primers respectively, the first strand cDNA of the variable region of the heavy chain and the light chain was synthesized under the action of M-MLV reverse transcriptase. Then, by PCR, SZ-63 heavy and light chain variable region genes were amplified. They were cloned into pUCl8 vector respectively and screened for positive clones. Nucleotide sequence analysis of ~ (32) P-labeled dideoxy terminator showed that the variable region of SZ-63 heavy chain was 354 bp long and encoded a polypeptide of 118 amino acids. The variable length of the light chain was 321 bp, encoding 107 amino acids . The SZ-63 variable region gene fragment was cloned with the human immunoglobulin γ1 heavy chain CHl and kappa light chain constant region genes by gene recombination technology to construct a phage plasmid and expressed in E. coli. The expressed chimeric antibody Fab fragment was soluble. The result of ELISA and Western blot showed that the expressed chimeric antibody could specifically bind with human fibrin. The expressed amount was about 225μg / L.