Zhang Xiao-Lei, Zhang Jin-Shun, Jia Xiao-Hui, Xu Yun-Peng, Zhang Ying, Wang Chun-Miao, Wang Yan, Lu Zhi-Min, Zhao Jian-Ling, Jia Tian-Jun
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2013 Dec;31(6):447-9.
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 ROP11 gene sequence and the Toxoplasma ROP11 gene from GenBank. Bioinformatics analysis showed that the ROP11 protein (Mr 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.
从刚地弓形虫RH株速殖子中提取总RNA。根据ROP11基因的编码序列(登录号:DQ077905)设计一对特异性引物,扩增ROP11基因的开放阅读框。RT-PCR产物用限制性内切酶EcoR I和Not I消化,然后连接到pGEX-6P-2载体中。重组质粒转入大肠杆菌XL-Blue。通过菌落PCR筛选阳性克隆,并经双酶切和测序进行鉴定。RT-PCR产物为1548 bp。通过菌落PCR和双酶切鉴定重组质粒。测序结果表明,获得的ROP11基因长度为1548 bp(登录号:KC456639)。获得的ROP11基因序列与GenBank中弓形虫ROP11基因的序列一致性较高(99%)。生物信息学分析表明,ROP11蛋白(Mr 57,020)由信号肽(氨基酸1-26)、12个保守结构域、一个丝氨酸/苏氨酸蛋白激酶催化结构域(氨基酸170-511)和两个潜在的N-糖基化位点组成。