刚地弓形虫ROP16基因的克隆及生物信息学分析  

Cloning and bioinformatic analysis of rhoptry protein 16 of Toxoplasma gondii

作　　者：刘楠[1] 郭玲玲[1] 张进顺[1] 张晓磊[1] 刘荣荣[1] 

机构地区：[1]河北北方学院病原生物学与免疫学研究所,河北张家口075000

出　　处：《中国病原生物学杂志》2016年第12期1108-1113,共6页Journal of Pathogen Biology

基　　金：河北北方学院校级重大课题(No.ZD201312)

摘　　要：目的构建刚地弓形虫棒状体蛋白16(TgROP16)真核重组表达载体,对TgROP16基因编码蛋白进行生物信息学分析。方法提取刚地弓形虫总RNA,根据TgROP16基因的开放阅读框架设计引物,采用逆转录PCR(RTPCR)扩增ROP16基因,经EcoR I、Not I酶切后连接入真核表达载体pVAX1中,连接产物转化大肠埃希菌(E.coli)XL1-Blue。提取阳性菌落质粒,进行PCR、双酶切鉴定及基因测序,对所获序列进行生物信息学分析。结果 TgROP基因RT-PCR扩增产物与预期一致,大小约为2 100bp。重组质粒经PCR及双酶切鉴定构建正确。测序显示获得的TgROP16基因片段为2 124bp,与GenBank已收录的弓形虫ROP16基因序列(登录号为GQ249093.1)比对,一致性为99.8%。预测其编码蛋白TgROP16为非跨膜分泌蛋白,含707个氨基酸,分子式为C3343H5339N967O1024S2,分子质量单位为76.1352ku,理论等电点为9.18;在TgROP16蛋白的二级结构中α-螺旋、延伸链、β-转角和无规则卷曲分别占比35.79%、14.29%、7.07%和42.86%;含多个可形成卷曲螺旋构象的区域与可成为磷酸化位点的氨基酸残基,存在信号肽序列,含39个B细胞抗原表位、24个CTL细胞抗原表位及33个Th细胞抗原表位。结论成功构建了pVAX1-ROP16真核表达重组质粒,生物信息学分析表明其编码蛋白TgROP16具有良好的抗原性及免疫原性,有望作为弓形虫疫苗候选分子。Objectives To construct a recombinant plasmid for eukaryotic expression of Toxoplasrna gondii rhoptry protein 16 （TgROP16） and to bioinformatically analyze the protein sequence encoded by the TgROP16 gene. Methods Total RNA was extracted from tachyzoites of T. gondii. Primers were designed in accordance with the open reading frame of the TgROP16 gene, Reverse-transcription PCR （RT-PCR） of the TgROP16 gene was performed. Tile RT-PCR product was digested with the restriction enzymes EcoR I and Not I and then ligated into the eukaryotic expression vector pVAX1. The recombinant plasmid was transformed into E. colt XL1-Blue. The plasmid extracted from positive clones was verified as correct with PCR, double restriction enzyme digestion, and sequencing. The protein sequence was bioin- formatiealiy analyzed. Results The RT-PCR product was about 2 100 bp in length, which is consistent with the expected size. The recombinant plasmid was verified as correct with PCR and double restriction enzyme digestion. Sequencing results indicated that the TgROP16 gene was 2 124 bp in length. The gene sequence is highly consistent （99.8%） with the TgROP16 gene in GenBank （Accession No. GQ249093. 1）. TgROP16 is predicted to code for a non-transmembrane and secretory protein consisting of 707 amino acids. Its molecular formula was Ca3343 H5339 N967 O1024 S2. Its molecular mass was 76. 1352ku, its theoretical isoelectric point was 9.18, and its average hydrophobicity index was 0.303. Alpha helices accounted for 35.79% of the secondary structure of TgROP16, extended strands accounted for 14.29%, beta turns ac counted for 7.07%, and random coils accounted for 42.86 %. TgROP16 has multiple areas that can form coiled-coil conformations and multiple amino acid residues that can serve as phosphorylation sites. TgROP16 has 1 signal peptide sequence, 39 potential B cell epitopes, 24 potential CTL epitopes, and 33 potential Th cell epitopes. Conclusion pVAXl was used as a carrier to construct the recombinant plasmid pVAX1-ROP16 for

关 键 词：刚地弓形虫 ROP16 真核表达 生物信息学分析 

分 类 号：R382.5[医药卫生—医学寄生虫学]