RIK蛋白的重组表达、多克隆抗体制备及其功能的初步研究

Recombinant expression, polyclonal antibody preparation and primary functional study of a new finding protein RIK

目的本研究从MHV68转化得到的小鼠B淋巴瘤细胞中筛选出一种功能尚未明确的蛋白质,暂时命名为RIK。通过对RIK基因克隆、表达和纯化并成功制备其多克隆抗体,并初步探讨其生物学功能,为该蛋白的进一步深入研究奠定基础。方法通过RT-PCR的方法检测B淋巴瘤细胞系和原代B细胞中的RIK m RNA水平,并扩增出RIK的基因片段,采用基因重组技术构建出RIK的原核表达载体,经诱导表达后,利用镍离子柱亲和纯化出RIK重组蛋白,免疫新西兰大耳兔,制备抗RIK多克隆抗体,ELISA和Western blot检测抗体灵敏度和特异性。最后,通过在过表达鼠源RIK的小鼠NIH3T12细胞中,进行MHV68病毒感染实验,初步确定其生物学功能。结果 RT-PCR检测发现RIK在B淋巴瘤细胞系中m RNA水平高于原代B细胞,并且成功构建人源和鼠源重组表达质粒p ET-28a(+)-h/m RIK,实现包涵体形式RIK蛋白的诱导表达纯化和抗体制备,SDS-PAGE和Western blot证实获得的抗RIK多克隆抗体与预期结果一致,能够特异地识别RIK蛋白。同时,研究发现,在小鼠NIH3T12细胞中过表达m RIK蛋白,能够有效的抑制MHV68对该细胞的感染。结论本研究成功实现了RIK的重组表达、抗体制备,并且发现RIK蛋白对于MHV68的感染具有十分有效的抑制作用,实现了对其功能的初步研究,并且为深入研究奠定了基础。

One new finding protein named as RIK is screened from our DNA microarray data in MHV68 transformed B lymphoma cells. This study focuses on cloning and expression of RIK gene fragment in E.coli, andpreparation of anti-RIK polyclonal antibody after purification of recombinant target protein, which will contribute tothe functional study of RIK in the future. Reverse transcription PCR was applied to obtain human and murine RIKgene fragments from human and mouse lymphoma cells, respectively. The RIK gene was cloned and constructed inprokaryotic expression vector through gene recombination technology. The expression of recombinant RIK wasinduced by IPTG and purified with Ni-NTA affinity chromatography. New Zealand rabbits were vaccinated with thepurified recombinant RIK and the anti-RIK polyclonal antibodies were prepared. The sensitivity and specificity ofpolyclonal antibody were analyzed by ELISA and Western blotting. RT-PCR results showed that the expression ofRIK in MHV68 transformed B lymphoma cells was notably higher than that in primary murine B cells. SDS-PAGEand Western blot results showed that RIK protein was expressed at the size of about 25 KD in the form of inclusionbody. ELISA and Western blot results showed that anti-RIK polyclonal antibodies prepared in New Zealand rabbitsexhibited high sensitivity and specificity. All the results indicated that RIK could express at high level in E.coli, andthe specific polyclonal antibodies against RIK were successfully obtained by rabbit vaccination and could be appliedto identify and analyze RIK expression. Furthermore, the study showed that overexpression of m RIK can effectivelyinhibit MHV68 infection in NIH3T12 cells, whichmakes a foundation for further functional study of RIK.