鲫鱼MBSP的原核表达、纯化及其多克隆抗体制备

Prokaryotic Expression，Purification of Myofibril-Bound Serine Proteinase from Crucian Carp（Carassius auratus） and Preparation of Its Polyclonal Antibody

DOI：10.3969/j.issn.1673-1689.2017.08.014

中文关键词: 肌原纤维结合型丝氨酸蛋白酶 原核表达 蛋白纯化 Western blot 多克隆抗体

英文关键词: myofibril-bound serine proteinase,prokaryotic expression,protein purification,western blot,polyclonal antibody

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中文摘要:

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。

英文摘要:

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.

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