马泰勒虫棒状体颈部蛋白5基因的克隆与原核表达

Cloning and prokaryotic expression of Theileria equi rhoptry neck protein 5 gene

从马泰勒虫新疆分离株PCR扩增棒状体颈部蛋白5(ron5)基因,构建pGEX-4T-ron5表达载体,转化至大肠埃希菌(E.coli)BL21(DE3)进行原核表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物,以谷胱甘肽S-转移酶标签兔单抗(1:2000)和马泰勒虫感染马阳性血清(1:100)作为一抗,以辣根过氧化物酶标记的羊抗兔IgG和兔抗马IgG(1:5000)为二抗进行蛋白质免疫印迹(Western blotting)分析。马泰勒虫新疆分离株的ron5基因长1434 bp,提交GenBank获得的登录号为OP777492。序列比对结果显示,马泰勒虫新疆分离株ron5的核苷酸和氨基酸序列与马泰勒虫WA株(GenBank登录号XM 004833657)的序列一致性最高,分别为99.6%和99.2%。系统进化树分析结果显示,马泰勒虫新疆分离株与马泰勒虫WA株聚在同一支上,二者亲缘关系最近。SDS-PAGE分析结果显示,异丙基硫代半乳糖苷(IPTG)诱导3 h时,RON5的表达量最高;RON5的相对分子质量(Mr)约为79000,主要以包涵体形式表达。Western blotting分析结果显示,RON5可被谷胱甘肽S-转移酶标签抗体和马泰勒虫感染马血清识别,在Mr 79000处有明显条带。本研究克隆了ron5,表达的RON5具有较好的抗原性。

The rhoptry-neck protein 5(ron5)gene was PCR amplified from the Xinjiang isolates of Theileria equi.The prokaryotic expression vector of pGEX-4T-ron5 was constructed and transformed into Escherichia coli BL21(DE3)for prokaryotic expression.The expression products was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Western blotting analysis was performed using glutathione S-transferase labeled rabbit monoclonal antibody(1:2000)and horse serum infected with T.equi(1:100)as primary antibodies,and horseradish peroxidase labeled sheep anti-rabbit IgG and rabbit anti-horse IgG(1:5000)as secondary antibodies.The ron5 of the Xinjiang isolate of T.equi was 1434 bp,and the entry number obtained by GenBank was OP777492.Sequence comparison results showed that the nucleotide and amino acid sequences of ron5 of the Xinjiang isolate were the most consistent with those of T.equi WA strain(GenBank accession number XM 004833657),which were 99.6%and 99.2%,respectively.Phylogenetic tree analysis showed that the Xinjiang isolate and the T.equi WA strain were clustered on the same branch,with the most closely relation.SDS-PAGE analysis showed that the protein expression was the highest at 3 h induced by isopropyl thiogalactoside.The relative molecular mass(Mr)of RON5 is about 79000,which is mainly expressed in the form of inclusion bodies.Western blotting results showed that RON5 could be recognized by glutathione S-transferase labeled antibody and horse serum infected with T.equi,with an obvious band at Mr 79000.In this study,ron5 was successfully cloned,and the RON5 expressed had good antigenicity.