GST沉降技术验证弓形虫醛缩酶与肌动蛋白的相互作用

中国寄生虫学与寄生虫病杂志 ›› 2011, Vol. 29 ›› Issue (5): 8-363-367.

GST沉降技术验证弓形虫醛缩酶与肌动蛋白的相互作用

郑斌, 尹志奎, 何蔼, 李卓雅, 詹希美

1 新乡医学院寄生虫学教研室，新乡 453000；2 中山大学中山医学院寄生虫学教研室，广州 510080；
3 新乡医学院药学院，新乡 453000

出版日期:2011-10-30 发布日期:2012-09-27

Protein Interaction between Aldolase and Actin of Toxoplasma gondii by GST Pull-down

ZHENG Bin, YIN Zhi-Kui, HE Ai, LI Zhuo-Ya, ZHAN Xi-Mei

1 Department of Parasitology，Xinxiang Medical University，Xinxiang 453000，China; 2 Department of Parasitology，Zhongshan School of Medicine，Sun Yat-sen University，Guangzhou 510089，China; 3 College of Pharmacy，Xinxiang Medical University，Xinxiang 453000，China

Online:2011-10-30 Published:2012-09-27

摘要/Abstract

摘要： 目的通过GST沉降技术（GST pull-down）验证刚地弓形虫醛缩酶（aldolase）与肌动蛋白（actin）的相互作用。方法 PCR扩增弓形虫cDNA中aldolase 和actin基因，分别亚克隆至原核表达质粒pGEX?鄄4T?鄄1和pET30a，转化至大肠埃希菌BL21（DE3），1 mmol/L异丙基?鄄β?鄄D?鄄硫代半乳糖苷（IPTG）诱导表达，亲和层析法纯化表达产物。采用腹部皮内多点注射免疫SD大鼠15只，首次免疫Actin-His6蛋白量为200 μg/只，第2次起免疫蛋白量为100 μg/只，共免疫4次，每次间隔7 d，末次免疫后5 d收集心脏血，制备Actin-His6抗血清。以纯化的GST-Aldolase蛋白作为探针蛋白与Actin-His6蛋白液进行GST沉降实验，实验产物进行十二烷基硫酸钠?鄄聚丙烯酰胺凝胶电泳（SDS?鄄PAGE）和蛋白质印迹（Western blotting）分析。结果获得了弓形虫aldolase和actin基因序列，构建了相应的原核表达载体。表达并纯化了GST-Aldolase和Actin-His6蛋白。Actin-His6蛋白免疫SD大鼠后获得其抗血清，经抗体亲和纯化柱纯化，获得Actin-His6多克隆抗体。SDS-PAGE和Western blotting 结果显示，GST沉降实验产物中的蛋白条带可被Aldolase-His6多克隆抗体和Actin-His6多克隆抗体识别。结论弓形虫醛缩酶与肌动蛋白存在相互作用。

关键词: 刚地弓形虫, 醛缩酶, 肌动蛋白, GST 沉降技术, 蛋白相互作用

Abstract: Objective To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down. Methods The aldolase and actin genes were obtained from cDNA library by PCR amplification，and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 μg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 μg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein，then incubated with Actin-His6，and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting. Results The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1，actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody. Conclusion Aldolase interacts with Actin of Toxoplasma gondii.

Key words: Toxoplasma gondii, Aldolase, Actin, GST pull-down, Protein interaction

郑斌, 尹志奎, 何蔼, 李卓雅, 詹希美. GST沉降技术验证弓形虫醛缩酶与肌动蛋白的相互作用[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(5): 8-363-367.

ZHENG Bin, YIN Zhi-Kui, HE Ai, LI Zhuo-Ya, ZHAN Xi-Mei. Protein Interaction between Aldolase and Actin of Toxoplasma gondii by GST Pull-down[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2011, 29(5): 8-363-367.

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