马链球菌兽疫亚种中国株的类M蛋白基因抗原表位片段的克隆与表达

目的　构建马链球菌兽疫亚种 (Streptococcusequisubsp zooepidemicus)中国株的类M蛋白基因抗原表位片段的重组表达质粒 (pET -Szp) ,并检测表达产物的免疫反应性。 方法　根据GenBank登录的马链球菌兽疫亚种纽约分离株w6 0类M蛋白基因序列设计和合成引物 ,以该菌中国分离株ATCC35 2 4 6的基因组DNA为模板 ,扩增类M蛋白基因 5’端第 5 83～ 10 6 8bp片段 ,并按正确的阅读框架定向克隆到表达载体pET - 32a(+)中 ,将重组质粒转化大肠杆菌BL2 1株 ,用IPTG诱导表达 ,并通过SDS -PAGE和免疫印迹对表达蛋白进行初步分析。结果　扩增出 4 86bp的马链球菌兽疫亚种ATCC35 2 4 6的类M蛋白基因片段 ,该基因在大肠杆菌表达系统中经诱导 ,得到分子量为 5 0 0 0 0的表达产物 ,免疫印迹表明该产物具有特异的免疫反应性。结论　成功构建了表达马链球菌兽疫亚种中国株类M蛋白片段的重组表达质粒 ,并实现在大肠杆菌中表达 ,为重组类M蛋白亚单位疫苗的研制奠定了基础。

Cloning and expression of the gene fragment encoding for the epitopes of M-like protein from the Chinese strains of Streptococcus equi subsp.zooepidemicus

To construct a recombinant plasmid containing the gene fragment encoding the epitopes of M-like protein from the Chinese strains of Streptococcus equi subsp. zooepidemicus and to determine the immunoreactivity of the expression products,one pair of primers was designed according to the gene sequences of the M-like protein of w60 strain isolated from New York,and the partial length of sequences of M-like protein was amplified from genomic DNA of ATCC35246 strain.Then,the amplified products were inserted into expression vector pET-32a( ),and the recombinant plasmid was transformed into E.coli BL 21 under the induction with IPTG.The expression products were analyzed by SDS-PAGE and Western blot analysis.The results showed that the length of the amplified fragment of the M-like protein gene was 486 bp,and the molecular weight of the fusion protein that was expressed by the recombinant plasmid in E.coli was 50,000.This product showed specific immunoreactivity with rabbit antiserum against ATCC35246 strain.These results could provide for the foundation for the development of the recombinant M-like protein subunit vaccines.