马泰勒虫RON2基因的原核表达及生物信息学分析

Prokaryotic expression and bioinformatics analysis of RON2 gene in Theileria equi

【目的】对马泰勒虫棒状体颈部蛋白2(RON2)进行原核表达和生物信息学分析,为后续蛋白互作试验提供材料基础。【方法】以马泰勒虫新疆分离株为研究对象,克隆其RON2基因,并与GenBank中已知的马泰勒虫美国WA株基因组数据和顶复门原虫RON2基因序列本地数据库进行BLAST比对,确定其进化关系。构建重组表达载体pET-32a-RON2,融合表达重组蛋白,对RON2基因编码蛋白进行生物信息学分析。【结果】马泰勒虫新疆株RON2基因(NCBI登录号MT939257)长3920 bp,高度保守,与顶复门原虫RON2基因核苷酸和氨基酸序列的相似性分别为31.2%~82.7%和23.8%~79.5%。成功构建原核表达重组质粒,获得的RON2融合重组蛋白分子质量为26 ku,为包涵体蛋白。生物信息学分析表明,RON2蛋白是一种碱性、不稳定亲水性跨膜蛋白,二级结构以α螺旋为主,亲水性较差,抗原性较弱,与其他顶复门原虫RON2蛋白具有相似的三级空间特殊结构和氨基酸序列,且可能与其他入侵蛋白存在相互作用关系。【结论】获得马泰勒虫新疆株RON2基因,诱导表达获得部分RON2融合重组蛋白,该蛋白存在与其他入侵相关蛋白相互作用的位点,可能参与了虫体入侵宿主细胞的过程。

【Objective】The prokaryotic expression and bioinformatics analysis of rhoptry neck protein 2(RON2)of Theileria equi were conducted to provide basis for further exploration of its role protein-protein interaction.【Method】The RON2 gene of T.equi strain from Xinjiang was cloned using the known genomic data of American WA strain of T.equi in GenBank and the RON2 gene sequence of Apicomplexan in local database.The BLAST alignments of RON2 gene were carried out,and the phylogenetic analysis was proceeded.Then,the recombinant expression vector pET-32a-RON2 was constructed,the fusion expression recombinant protein was expressed,and the protein encoded by RON2 gene was analyzed by bioinforma-tics.【Result】The RON2 gene(NCBI accession number MT939257)of the T.equi strain in this study was 3920 bp in length.The nucleotide and amino acid sequence similarity with the RON2 genes of Apicom-plexa were 31.2%-82.7%and 23.8%-79.5%,respectively,indicating that RON2 gene was highly conserved.Prokaryotic expression recombinant plasmid was successfully constructed,and the recombinant protein of RON2 fusion with a molecular weight of 26 ku was obtained,which was an inclusion body protein.Bioinformatics analysis showed that RON2 protein is alkaline,unstable and hydrophilic transmembrane protein.Its secondary structure was mainly alpha helix with poor hydrophilicity and weak antigenicity,and it had similar special three-level space structure and amino acid sequence with other Apicomplexus RON2 proteins.It may interact with other invasion proteins.【Conclusion】The RON2 gene of the T.equi from Xinjiang was obtained,and part of its fusion recombinant protein was obtained by inducing expression.This protein had interaction sites with other invasion-related proteins and may be involved in parasite invasion into host cells.