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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。
Objective This study screened a protein that has not been elucidated from mouse B lymphoma cells transformed by MHV68, and named it RIK temporarily. The cloning, expression and purification of RIK gene and the successful preparation of its polyclonal antibody, and preliminary study of its biological function, laid the foundation for further study of RIK gene. Methods RIK m RNA levels in B lymphoma cell lines and primary B cells were detected by RT-PCR and the RIK gene fragments were amplified. The prokaryotic expression vector of RIK was constructed by gene recombination technique. After induced expression, The RIK recombinant protein was purified by nickel ion column and immunized New Zealand rabbits to prepare anti-RIK polyclonal antibody. The sensitivity and specificity of the antibody were detected by ELISA and Western blot. Finally, the MHV68 virus infection experiment was performed in mouse NIH3T12 cells overexpressing mouse RIK, and its biological function was initially determined. Results RT-PCR showed that the level of m RNA of RIK in B lymphoma cell lines was higher than that of primary B cells, and the recombinant plasmid p ET-28a (+) - h / m RIK was constructed successfully. The expression and purification of RIK protein in vivo and antibody preparation were confirmed. The anti-RIK polyclonal antibodies obtained by SDS-PAGE and Western blot were consistent with the expected results and could specifically recognize RIK protein. At the same time, the study found that in mice NIH3T12 cells overexpression of m RIK protein, can effectively inhibit MHV68 infection of the cells. Conclusions The recombinant expression and antibody preparation of RIK were successfully carried out in this study. It was found that RIK protein had a very effective inhibitory effect on MHV68 infection, and a preliminary study of its function was established, which laid the foundation for further study.