[刚地弓形虫棒状体蛋白18（ROP18）的克隆、表达及免疫反应性分析]
[Cloning, expression and immunoreactivity analysis of rhoptry protein 18 (rop18) from Toxoplasma gondii].
作者信息
Zhang Ying, Zhang Jin-Shun, Jia Xiao-Hui, Jia Tian-Jun, Lu Zhi-Min, Zhou Fang
机构信息
Institute of Pathogen Microbes and Immunology, Hebei North University, Zhangjiakou, 075000, China.
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Dec 30;30(6):446-9.
OBJECTIVE
To clone and express rhoptry protein 18 (ROP18) gene of Toxoplasma gondii, and analyze its immunoreactivity.
METHODS
The genomic DNA was extracted from T. gondii (RH strain) tachyzoites. TgROP18 gene was amplified by PCR, and cloned into pET30a (+) vector. The constructed pET30a (+)-TgROP18 was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed through SDS-PAGE, and identified by Western blotting with mouse anti-T. gondii serum.
RESULTS
The TgROP18 gene was about 1 665 bp in length and encoded for a protein of 544 amino acid residues and the former 47 amino acids consisted signal peptide sequences. PCR, enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET30a (+)-TgROP18 was constructed. Bacteria containing recombinant plasmid pET30a (+)-TgROP18 expressed a soluble protein of His-TgROP18 (M, 59 800) after being induced with IPTG. His-TgROP18 reacted positively with mouse anti-T. gondii serum by Western blotting analysis.
CONCLUSION
The soluble His-TgROP18 protein shows certain immunoreactivity.
目的
克隆并表达刚地弓形虫棒状体蛋白18（ROP18）基因，并分析其免疫反应性。
方法
从刚地弓形虫（RH株）速殖子中提取基因组DNA。通过PCR扩增TgROP18基因，并克隆到pET30a（+）载体中。将构建好的pET30a（+）-TgROP18转化到大肠杆菌BL21（DE3）中，然后用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达该蛋白。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白，并用小鼠抗弓形虫血清进行蛋白质印迹鉴定。
结果
TgROP18基因长度约为1 665 bp，编码一个由544个氨基酸残基组成的蛋白质，前47个氨基酸为信号肽序列。PCR、酶切和DNA测序证实构建了重组质粒pET30a（+）-TgROP18。含有重组质粒pET30a（+）-TgROP18的细菌经IPTG诱导后表达出一种可溶性的His-TgROP18蛋白（分子量59 800）。蛋白质印迹分析显示His-TgROP18与小鼠抗弓形虫血清呈阳性反应。
结论
可溶性His-TgROP18蛋白具有一定的免疫反应性。
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