摘要
目的原核表达并纯化埃可病毒9型(ECHO9)VP1蛋白,免疫兔子制备多克隆抗体,鉴定重组蛋白的免疫活性,为ECHO9血清学检测试剂和疫苗研究提供依据。方法 PCR方法扩增VP1基因,构建原核表达质粒p ET28a-VP1并转化到大肠杆菌(E.coli BL21),诱导表达并纯化VP1重组蛋白,免疫兔子制备抗VP1多克隆抗体,ELISA检测抗VP1抗体效价,Western blot鉴定抗体特异性。病毒中和试验鉴定抗体中和ECHO9病毒的能力。结果 VP1重组蛋白在大肠杆菌BL21中高效表达,制备的抗VP1抗体效价为1∶105。Western blot检测结果显示,抗VP1多克隆抗体可以识别原核表达的VP1蛋白。中和试验显示抗体对ECHO9病毒有中和作用,其效价为1∶10。结论本研究成功原核表达了ECHO9的VP1蛋白并制备出抗VP1多克隆抗体,有助于ECHO9的临床诊断、疫苗开发和分子病毒学研究。
Objective To express and purify the recombinant capsid protein VP1 of echovirus type 9（ ECHO9）,and prepare the corresponding VP1-specific polyclonal antibody and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. Methods The VP1 gene was amplified by PCR and cloned into vector of p ET28 a to make p ET28a-VP1 for the prokaryotic expression of VP1. The recombinant VP1 protein was expressed in E. coli BL21 harboring p ET28a-VP1,purified from inclusion bodies,renatured,and subsequently used to immunize rabbits. The resultant antisera were evaluated for anti-VP1 titer,bingding capacity and specificity by ELISA,Western blot assays. Through virus neutralization test,identify the ability to neutralize Echo9 of antisera. Results Recombination protein VP1 was efficiently produced in E. coli. Immunization of rabbits with recombinant elicited high-titer（1∶105） VP1-specific antibodies. Western blot analysis showed the resultant anti-VP1 sera reacted with E. coli expressed VP1. Through virus neutralization test,we find the antisera can neutralize Echo9,the titer of 1∶10. Conclusion The recombinant VP1 and the corresponding polyclonal antbodies can be used to identify and characterize ECHO9,which could be useful for developing diagnose reagent or vaccine of ECHO9.
出处
《中国卫生检验杂志》
CAS
2015年第7期951-953,965,共4页
Chinese Journal of Health Laboratory Technology
基金
杭州市科技发展计划项目(20120633B22)
