中文摘要：

绵羊肺炎支原体（Mycoplasma ovipneumoniae,MO）可引起绵羊（Ovis aries）及山羊（Capra hircus）的支原体性肺炎,分布广泛,危害严重。本实验在课题组前期完成MO Y98株基因组测序的基础上,通过基因注释、PCR等获得了该菌株可能的溶血素（hemolysin,hly A）基因;依据大肠杆菌（Escherichia coli）优势密码子对hly A基因进行优化,构建优化后hly A原核表达质粒p ET32a-hly A;将该质粒转入大肠杆菌BL21（DE3）p Lys S中,获得了相应的重组菌株,并对重组菌株进行诱导表达;采用SDS-聚丙烯酰胺凝胶电泳（SDS polyacrylamide gel electrophoresis,SDS-PAGE）和Western blot对表达的重组蛋白进行鉴定,并经Ni2＋金属螯合柱纯化。克隆了MO hly A基因序列（Gen Bank登录号：KT598390）,片段大小为375 bp,编码125个氨基酸。优化后密码子适应指数为0.91,目的基因适于在大肠杆菌中表达。重组融合蛋白多以包涵体的形式存在,其相对分子质量约为31.6 k D,与预期大小一致。Western blot结果显示,上清中纯化出的目的蛋白能与一抗发生反应,重组蛋白特异性较好。溶血实验表明,当纯化后蛋白浓度稀释到0.125μg/m L时,溶解红细胞的能力已经下降。该研究结果为后续绵羊肺炎支原体致病性研究提供了基础资料。

英文摘要：

Mycoplasma ovipneumoniae（MO） can lead to the atypical pneumonia for both sheep（Ovis aries）and goats（Capra hircus）, which is wide- spreading and can cause serious harm. Based on the complete sequence of MO Y98 strain by our group, a potential hemolysin gene（hly A） was obtained by using gene annotation and PCR method. In the present study, hly A gene, which derived from MO Y98, was modified by using preference codons of Escherichia coli. And then prokaryotic expression vector（p ET32a- hly A） was constructed. p ET32a-hly A was transformed into E. coli BL21（DE3） polys S, and induced for the expression of Hly A. Hly A recombinant protein was visualized by SDS polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot method, and purified by using Ni2 ＋metal chelate column. The results showed that the hly gene（Gen Bank No.KT598390） of MO Y98 strain contained 375 bp, encoding a polypeptide of 125 amino acid residues. After optimization, the codon adaptation index was 0.91, the modified hly A is suitable for expression in E. coli. Recombinant fusion proteins were mainly existed in the form of inclusion body. Expressed Hly Aprotein was about 31.6 k D, consistenting with the expected size. The results of Western blot analysis showed that supernatant of purified protein could react with the primary antibody, indicating that Hly A proved an efficacious immunological reactivity. When purified recombinant Hly A protein concentration was diluted to0.125 μg/m L, the ability of dissolving red blood cells had declined. This research contributes to the study of hymolysin pathogenicity in MO.