摘要
目的制备细粒棘球绦虫PI3KP110亚基酪氨酸激酶区（EgPI3KP110RTK）蛋白的多克隆抗体,为进一步研究EgPI3KP110蛋白的功能提供检测抗体。方法将EgPI3KP110RTK（1-352aa）基因序列克隆至原核表达载体pGEX-4T-1,转化E.coli ROSETTA（DE3）株,IPTG诱导蛋白表达,以GST亲和层析纯化EgPI3K P110RTK蛋白,免疫家兔,收集免疫血清,采用ELISA法测定抗体效价,Western blotting检测所制备抗体与EgPI3KP110RTK蛋白的反应性。结果经0.8mmol/L IPTG诱导,表达出约66ku的EgPI3KP110RTK重组蛋白,制备获得效价在5.12×10^5以上的多克隆抗体,该抗体能够与EgPI3KP110RTK重组蛋白发生特异性反应。结论成功制备获得兔抗EgPI3KP110亚基多克隆抗体,为研究EgPI3K激酶在Eg中的功能和作为药物靶标等研究奠定基础。
Objective To generate the polyclonal antibody against the PI3 K P110 subunit protein of Echinococcus granulosus（Eg）for further study on the function of Eg PI3KP110 protein.Methods The expressing sequence of EgPI3KP110 subunit tyrosine kinase protein（1-352AA）was subcloned into the prokaryotic expression vector pGEX-4T-1and transformed into E.coli ROSETTA（DE3）strain.The recombinant protein expression was induced by 0.8mmol/L IPTG,followed by purification using GST affinity chromatography and vaccinated New Zealand rabbits.The titers and reactivity of antibody was evaluated by ELISA and Western blotting.Results The recombinant prokaryotic expression vector was successfully constructed,verified by restriction endonuclease digesting and DNA sequencing.Then,recombinant GST-Eg PI3KP110 awas successfully expressed in E.coli ROSETTA（DE3）after induction by0.8 mmol/L IPTG,the recombinant GST-EgPI3 K P110RTK protein was 66 KD.The rabbit was vaccinated with the GST-EgPI3 K P110RTK protein purified by affinity chromatography.ELISA showed that the titer of the anti-serum we′ve generated was above 5.12×10^5,and western-blotting result showed that the anti-serum could specifically recognize recombinant EgPI3 K P110RTK.Conclusion The polyclonal antibody against the EgPI3KP110 was successfully generated,which could lay a foundation for further study on the function of EgPI3 Kkinase.
出处
《新疆医科大学学报》
CAS
2016年第5期569-572,共4页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区包虫病基础医学重点实验室开放课题(XJDX0202-2013-10)
