鲫鱼MBSP的原核表达、纯化及其多克隆抗体制备

摘要

构建鲫鱼肌原纤维结合型丝氨酸蛋白酶(MBSP)的原核表达菌株Rosetta(pET28a-MBSP),获得重组MBSP,以制备MBSP多克隆抗体.将鲫鱼MBSP全长基因(MBSP)借助表达载体pET-28a构建重组表达菌株Rosetta(pET28a-MBSP),并进行诱导表达后获得重组MBSP.利用Ni-NTA agarose亲和层析柱纯化重组蛋白并进行质谱鉴定.以纯化后的重组MBSP作为抗原免疫新西兰兔,获得MBSP多克隆抗体.分别采用ELISA和Western blot技术测定其效价和特异性.诱导表达的重组蛋白经SDS-PAGE分析、Western blot检测及质谱鉴定,结果表明该蛋白质为重组MBSP,其相对分子质量约为28×103,与天然MBSP大小一致,主要以包涵体形式存在.将其免疫兔子后,从血清中获得了与MBSP发生特异性反应的高效价多克隆抗体.本研究中成功表达和纯化了重组MBSP蛋白,制备了高效价的特异性MBSP多克隆抗体,为MBSP的相关研究奠定基础.%An E.coli expression strain Rosetta (pET-28a-MBSP) of myofibril-bound serine proteinase (MBSP) from crucian carp (Carassius auratus) was constructed.The recombinant MBSP (rMBSP) was expressed and purified in order to prepare its polyclonal antibody,which would lay a foundation for further studies on the MBSP.The MBSP gene was transformed into Rosetta by sub-cloning it into pET-28a vector to construct the strain Rosetta(pET-28a-MBSP).The recombinant strain was induced to express MBSP by lactose.The recombinant protein was purified by Ni-NTA agarose affinity column chromatography,MALDI-TOF-MS identification,and then use as the antigen to immune the rabbits to prepare polyclonal antibody.Antibody titer was assayed by ELISA and specificity was detected by western blot.SDS-PAGE,western blot and MALDI-TOF-MS analysis showed that the recombinant protein was the recombinant MBSP (rMBSP) with molecular weight of approximately 28 ×103,which was similar to native MBSP from the skeletal muscle of crucian carp.And the rMBSP was expressed in prokaryotic expression system in mainly inclusion body.The serum with a high titer and specificity was obtained from immunized rabbit.The recombinant MBSP was expressed and purified successfully.The MBSP's polyclonal antibody with a high titer and specificity was obtained by immunization using the purified MBSP.