北京长白杂交猪IgGH链基因CH2-CH3的克隆、序列分析及表达质粒构建

Cloning and Prokaryotic Expression of CH2-CH3 Gene of Beijing Chang-bai Crossbred Swine IgG Heavy Chain

根据GenBank中注册的猪IgG H链基因cDNA CH2 CH3序列(SSU03780),设计了含KpnⅠ和HindⅢ酶切位点的一对引物,采用反转录-聚合酶链式反应(RT-PCR)法从北京长白杂交猪肠系膜淋巴结细胞扩增猪IgG H链基因CH2 CH3序列.将PCR产物纯化回收后,插入到含LacZ基因的克隆载体pGEM-T Easy中,转化大肠杆菌JM 109感受态细胞,经KpnⅠ和HindⅢ双酶切及PCR鉴定,筛选出阳性克隆.将所克隆的目的片断亚克隆到原核表达载体pQE 30,构建重组质粒pQE 30/PIgG CH2 CH3.将重组表达质粒转化大肠杆菌JM 109感受态细胞,筛选阳性克隆,经IPTG诱导表达后,进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析.结果表明,本研究克隆了猪IgG H链基因CH2 CH3序列,全长663 bp,编码221个氨基酸,利用Gentyx version 6.0软件将CH2 CH3基因的核苷酸序列与GenBank中的参考序列比较,其核苷酸序列同源率为99.551%,推导的氨基酸序列同源率为100%.将pQE 30/PIgG CH2 CH3转化JM 109感受态细胞,表达的融合蛋白相对分子质量约为26.95 ku,经IPTG诱导2 h,表达量约占菌体总蛋白41.5%.

According to the sequence of porcine IgG H chain gene CH2-CH3 sequence （SSU03780） in GenBank, a pair of primers containing KpnI and Hind Ⅲ restriction sites respectively were designed. The constant region of IgG heavy chain of porcine CH2 and CH3 domains were cloned from Beijing Changbai crossbred swine mesentery lymph node by RT- PCR. After being purified, the PCR product was inserted into the pGEM - T Easy vector. The recombinant pGEM - T Easy/PIgG CH2-CH3 plasmid was digested by double enzymes （ Kpn Ⅰand Hind Ⅲ ）. The DNA fragment of interest was subcloned into the expression vector pQE 30, which contained the T5 promoter and the N-terminal 6 consecutive histone residues. The recombinant plasmid pQE 30/PIgG CH2-CH3 was identified by restriction enzyme analysis （ Kpn Ⅰand Hind Ⅲ） and the inserted gene was sequenced （Boya Bio-teehnology Co. LTD, Shanghai）. The engineering JM 109 strain containing pQE 30/PIgG CH2-CH3 was induced by IPTG and SDS-PAGE analysis was carried out to identify the expressed protein. Results show that the IgG CH2-CH3 gene of Porcine is composed of 663 bp nucleotides encoding 221 amino acid residues, which is in correspondence to the predicted result. Three nucleotides changes were found in the igG CH2-CH3 gene of porcine and the homology ratios of nucleotides and amino acids were 99.551% and 100% respectively comparing with the sequence of IgG gene of porcine in GenBank.