GST沉降技术验证弓形虫醛缩酶与肌动蛋白的相互作用

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

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-4T-1和pET30a,转化至大肠埃希菌BL21(DE3),1 mmol/L异丙基-β-D-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200μg/只,第2次起免疫蛋白量为100μg/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-聚丙烯酰胺凝胶电泳(SDS-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。

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.