驽巴贝虫BC-48基因的克隆及其在大肠杆菌中的表达

Cloning of BC-48 gene of Babesia caballi Jilin strain and its expression in Escherichia coli

采用PCR方法扩增驽巴贝虫吉林分离株BC-48基因片段,将扩增产物与pGEM-T Easy载体连接,重组质粒经PCR、单酶切鉴定后测序;构建BC-48的重组pGEX-4T-2表达载体,经IPTG诱导表达后,进行SDS-PAGE、Western-blotting分析。结果显示,克隆的BC-48基因片段长610 bp,含有一个570 bp的开放阅读框,编码189个氨基酸,与GenBank中USDA株(U46551)的同源性为96.7%;表达的融合蛋白为45 ku,能被驽巴贝虫阳性血清识别;表明该融合蛋白具有较好的反应原性。

BC-48 gene of Babesia caballi of Jilin strain was amplified by PCR and ligated into pGEM-T Easy vector to construct recombinant plasmid. The recombinant plasmid was certified by PCR, endonuclease dig/estion and sequencing. A prokaryotic expression plasmid was constructed by inserting the BC-48 gene into pGEX-4T-2 vector. Expressed protein induced by IPTG was analyzed by SDS-PAGE and Western-blotting. The BC-48 cDNA was 610 bp in length with a 570 bp ORF encoding 189 amino acids. Similarity of nucleotide sequence of the BC-48 gene with the sequence from GenBank（U46551）was 96.7%. The 45 ku expressed fusion protein was recognized by positive serum against Babesia caballi ,showing that the fusion protein have strong reactogenicity.