刚地弓形虫棒状体蛋白17基因的克隆、表达及抗原性分析（英文）

中国寄生虫学与寄生虫病杂志 ›› 2012, Vol. 30 ›› Issue (1): 5-27-31.

刚地弓形虫棒状体蛋白17基因的克隆、表达及抗原性分析（英文）

王海龙1，殷丽天2，孟晓丽1，申金雁1，刘红丽1，殷国荣1 *

1 山西医科大学寄生虫学教研室，太原 030001；2 山西医科大学生理学系，太原 030001

出版日期:2012-02-29 发布日期:2012-09-28

Gene-cloning， Expression and Antigenicity Analysis of Rhoptry Protein 17 of Toxoplasma gondii

WANG Hai-long1，YIN Li-tian2，MENG Xiao-li1，SHEN Jin-yan1，LIU Hong-li1，YIN Guo-rong1 *

1 Department of Parasitology，Shanxi Medical University，Taiyuan 030001，China；
2 Department of Physiology，Shanxi Medical University，Taiyuan 030001，China

Online:2012-02-29 Published:2012-09-28

摘要/Abstract

摘要： 目的克隆、表达刚地弓形虫（Toxoplasma gondii） RH株棒状体蛋白17（TgROP17）基因，并分析其抗原性。方法制备弓形虫RH株速殖子总RNA，根据TgROP17基因全长编码序列（GenBank登录号为AM075203.1）的开放阅读框设计引物并进行逆转录PCR（RT-PCR）扩增，扩增产物经双酶切后连接入pGEX-6P-1载体，重组质粒转化大肠埃希菌（E. coli）DH5α，阳性菌落经PCR和双酶切鉴定，并测序。将重组质粒pGEX-6P-1-TgROP17转化至E. coli Rosetta（DE3）并加入异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，十二烷基磺酸钠?鄄聚丙烯酰胺凝胶电泳（SDS-PAGE）结合考马斯亮蓝染色检测表达产物。分别以兔抗弓形虫血清和抗谷胱甘肽S转移酶（GST）标签抗体为一抗，采用蛋白质印迹（Western blotting）分析重组蛋白及其抗原性。结果 RT?鄄PCR扩增产物约为1 850 bp。菌落PCR、双酶切及测序结果显示重组质粒pGEX-6P-1-TgROP17构建成功。SDS-PAGE结果显示，经IPTG诱导获得相对分子质量（M_r）约96 000的可溶性重组蛋白。Western blotting结果表明，诱导表达的蛋白质为带GST标签的重组蛋白，且能被兔抗弓形虫血清识别。结论获得刚地弓形虫重组ROP17蛋白，且具有抗原性。

关键词: , 刚地弓形虫；棒状体蛋白17；原核表达；抗原性

Abstract: Objective To clone and express the rhoptry protein 17 （ROP17） gene of RH strain of Toxoplasma gondii，analyze the antigenicity of recombinant protein. Methods Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgROP17 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgROP17 gene （GenBank accession No. AM075203.1），the product of RT-PCR was digested with double restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-TgROP17 plasmid was transferred into E. coli DH5α and the positive clones were selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The constructed pGEX-6P-1-TgROP17 was transformed into E. coli Rosetta （DE3） and induced with IPTG for expression. The expression products were analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with GST primary antibody and rabbit anti-T. gondii serum was used to confirm the expression of GST-ROP17 and analyze its antigenic properties. Results The product of RT-PCR was with 1 850 bp. The recombinant plasmid pGEX-6P-1-TgROP17 was confirmed by colony-PCR，double restriction enzyme digestion and sequencing. A soluble recombinant protein with relative molecular weight of 96 000 was analyzed by SDS-PAGE followed by Coomassie blue staining. The GST tag in GST-ROP17 and the antigenicity of ROP17 were detected efficiently by Western blotting with the GST primary antibody and with the prepared antiserum against T. gondii, respectively. Conclusion The recombinant GST-ROP17 protein has been produced in E. coli and shows specific antigenicity.

Key words: Toxoplasma gondii, Rhoptry protein 17, Prokaryotic expression, Antigenicity

王海龙1，殷丽天2，孟晓丽1，申金雁1，刘红丽1，殷国荣1 *. 刚地弓形虫棒状体蛋白17基因的克隆、表达及抗原性分析（英文）[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(1): 5-27-31.

WANG Hai-long1，YIN Li-tian2，MENG Xiao-li1，SHEN Jin-yan1，LIU Hong-li1，YIN Guo-rong1 *. Gene-cloning， Expression and Antigenicity Analysis of Rhoptry Protein 17 of Toxoplasma gondii[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2012, 30(1): 5-27-31.

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