羊布鲁菌陕西分离株OMP19基因的克隆、序列分析及原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of OMP19 Gene of Goat Brucellain Shaanxi Province

对羊布鲁菌(Brucella)陕西分离株OMP19基因进行克隆、序列分析及原核表达。利用GenBank中收录的布鲁菌(KT229642.1)OMP19基因序列,设计合成引物,应用PCR技术扩增OMP19基因,并将OMP19基因连接到原核表达载体pET-28a中,构建重组质粒pET28a-OMP19,转化到BL21感受态细胞进行诱导表达,通过SDS-PAGE和Western blot法分析。结果克隆了羊布鲁菌陕西分离株OMP19全基因序列,核苷酸序列分析表明,陕西分离株OMP19基因与国内外已报道的羊布鲁菌核苷酸同源性超过98%,氨基酸同源性超过98%。OMP19重组菌经诱导表达约为19ku的重组蛋白,该蛋白能与本实验室鉴定保存的阳性血清特异性结合,反应原性良好。为羊布鲁菌陕西分离株分子生物学特性研究提供资料,为进一步进行基因工程疫苗研制及ELISA试剂盒抗体检测提供了基础。

Cloning, sequence analysis and prokaryotic expression of OMP19 gene of Brucella Shaanxi isolate were carried out. OMP19 gene was amplified by PCR using the OMP19 gene sequence of Brucella （KT229642.1） in GenBank.The OMP19 gene was ligated into the prokaryotic expression vector pET-28a and the recombinant plasmid was successfully constructed. PET28a-OMP19 was transformed into BL21 competent cells to induce expression.SDS-PAGE and Western blot analysis were used for detecting the expression.The OMP19 gene of Shaanxi isolate was successfully cloned.The nucleotide sequence of OMP19 gene of Shaanxi isolate was found to be over 98% similarity to that reported in China and abroad.The amino acid similarity was more than 98%.OMP19 recombinant protein was induced to express about 19 ku recombinant protein, the protein can be identified with positive serum, showing good specificity and reactivity.This study provided the data for the study on the molecular biological characteristics of Brucella isolates in Shaanxi province,and provided the basis for further genetic engineering vaccine development and antibody detection by ELISA kits.