抗盐酸克伦特罗单链抗体基因的克隆、序列分析及表达

Cloning nucleotide sequencing and configuration forecast of ScFv against clenbuterol and expression

提取分泌抗盐酸克伦特罗单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,用一段柔性肽链-(G4S)3将VH和VL连接成ScFv。测序后经NCBI Blast分析,所得ScFv具有重组功能性鼠抗体可变区基因的特征。将所得目的基因与pET-22b(+)连接,转化E.coli BL21,用IPTG诱导表达,表达蛋白经SDS-PAGE分析,37℃培养5 h表达量较大,25℃诱导表达以可溶性为主。

The total RNA of the hybridoma cell,secreting monoclonal antibody of anti-Clenbuterol, was abstracted and the ScFv was gained through linking the fragments with a peptide bond-（G4S）3 after the DNA fragments of VH and VL, is obtained with the technique of RT-PCR. The ScFv was cloned in the vector of pMD18-T. After sequenced by Shanghai Sagon, the results were analyzed on the Blast of National Center for Biotechnology Information. The ScFv has the characters of variable domain of reconstructed functional mouse antibody. The DNA fragment and pET-22b （＋） are linked with T4 DNA Ligase. The linked system was transformed in E. coli BL21 and inducted with IPTG. The expressed protein was analyzed through 12%SDS-PAGE. When inducted for 5 h in the temperature of 37 C, the protein is most. Under the temperature of 25 C the protein is mainly expressed with soluble protein.