肠道病毒71型2C蛋白原核表达及抗体制备

Prokaryotic Expression and Antibody Preparation of Enterovirus Type 71 Protein 2C

肠道病毒71型(EV71)的非结构蛋白2C(P2C)在病毒复制周期中起着重要的作用,制备P2C的特异性抗体,对研究P2C的生物学功能以及EV71与宿主相互作用的具体机制有非常重要的意义。实验将2C基因克隆到原核表达载体p ET-28a(+)上,在大肠埃希菌BL21(DE3)中表达出重组蛋白r P2C,进一步优化原核表达条件,在温度为30℃,诱导剂IPTG浓度为1 mmol/L时,蛋白表达量最高,且主要以包涵体形式存在。直接将获得的r P2C通过SDS-PAGE分离后免疫新西兰兔,制备EV71病毒P2C的兔多克隆抗体。通过Western blot检测,该抗体在110 000稀释比例下仍能很好地识别原核表达的r P2C。同时该抗体也能很好地检测到EV71感染RD细胞中的P2C。因此,实验制备出的抗P2C抗体特异性强、效价高,为后续P2C功能的研究以及EV71病毒检测提供了良好的材料。

Nonstructural protein 2C（ P2C） of Enterovirus 71（ EV71） plays an important role in viral duplication cycle. Preparation of P2 C specific antibody has very important significance to study the biological function of P2 C and interaction mechanism between EV71 and its host. The 2C gene was cloned into the prokaryotic expression vector p ET-28a（ ＋）. The recombinant P2 C was expressed in E. coli BL21 with the plasmid p ET28a-2C. After optimization it was comfirmed that under 30 ℃ and inducer concentration at 1 m mol / L IPTG,the exprssion of P2 C reached to the highest and mainly existed in a form of inclusion body. The obtained P2 C was immunized into New Zealand white rabbits after sepratated by SDS-PAGE and prepare polyclonal antibody. It was tested through western blot that the antibody still could be well identified the prokaryotic expressed P2 C even diluted by 110 000. Thus it can be seen that antiP2 C antibody prepared in this experiment had high specificity,with high valence; it provided good material for the following up studies on the function of P2 C and the detection of EV71 virus.