刚地弓形虫ROP11基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Rhoptry Protein 11 of Toxoplasma gondii

提取刚地弓形虫（Toxoplasmagondii）RH株速殖子总RNA，根据棒状体蛋白11（ROP11全长编码序列（登录号为DQ077905）的开放阅读框设计引物并进行逆转录PCR（RT-PCR）扩增，PCR产物经EcoRI和NotI酶切后与原核表达载体pGEX-6P-2连接，重组质粒转化大肠埃希菌（E．coli）XL-Blue，阳性菌落经PCR和双酶切鉴定，并测序。对所得序列进行生物信息学分析。结果显示，RT-PCR扩增产物约为1500bp。菌落PCR及双酶切结果正确。测序结果显示，获得的ROP11基因片段为1548bp（登录号为KC456639），与GenBank上已有的弓形虫ROP11序列相比，序列一致性为99％。生物信息学分析发现，ROP11编码蛋白质的预期相对分子质量为M57020，包括有12个保守结构区域，其前26个氨基酸残基构成信号肽，丝氨酸／苏氨酸蛋白激酶催化区域位于170～511氨基酸，且有2个潜在的N-糖基化位点。

Total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii. The open reading frame of ROP11 gene was amplified by using a pair of specific primers designed according to the coding sequence of ROP11 gene （Accession No. DQ077905）. The RT-PCR product was digested by restriction enzyme EcoR I and Not I, and then ligated into a pGEX-6P-2 vector. The recombinant plasmid was transferred into E. coli XL-Blue. The positive clones was selected by colony PCR, and confirmed by the double restriction enzyme digestion and sequencing. The RT-PCR product was 1 548 bp. The recombinant plasmid was confirmed by colony PCR and double restriction enzyme digestion. Sequencing results showed that the obtained ROP11 gene was 1 548 bp （Accession No. KC456639）. There was a high sequence consistency （99%） between the obtained ROPll gene sequence and the Toxoplasma ROPll gene from GenBank. Bioinformatics analysis showed that the ROPll protein （M, 57 020） consisted of the signal peptide （amino acids 1-26）, 12 conservative domains, a serine/threonine protein kinase catalytic domain （amino acids 170-511）, and two potential N- glycosylation sites.