人Shisa样蛋白1(SHISAL1)抗原表位区预测及其小鼠多克隆抗体的制备

Prediction of epitope region and preparation of mouse polyclonal antibody of human Shisa-like protein 1(SHISAL1)

目的优化人Shisa样蛋白1(SHISAL1)抗原后制备小鼠抗多克隆抗体,并鉴定抗体特异性.方法利用生物信息学方法预测SHISAL1蛋白的抗原表位区,选取SHISAL1蛋白第28~97位氨基酸残基组成的多肽作为抗原,并命名为(SHISAL1-N);利用分子克隆技术,将其编码序列克隆后插入pET-28a载体中构建重组质粒pET28a-SHISAL1-N.然后将pET28a-SHISAL1-N和已构建的pET28a-SHISAL1全长重组质粒转化BL21(DE3),用异丙基-β-D硫代吡喃半乳糖苷(IPTG)诱导蛋白表达.两种蛋白纯化后分别免疫雌性昆明小鼠,Western blot法、免疫沉淀及免疫荧光细胞化学染色法观察两种抗体的特异性与灵敏性.结果成功构建pET28a-SHISAL1-N重组质粒,诱导两种融合蛋白表达,获得两种类型的SHISAL1小鼠多克隆抗体.Western blot结果表明以SHISAL1为免疫原所制备的抗体特异性和灵敏性较差,而以SHISAL1-N免疫原所制备的抗体能特异性识别不同的细胞内源性的SHISAL1蛋白;免疫沉淀结果表明SHISAL1-N抗体可以结合肝癌细胞中的SHIISAL1蛋白;免疫荧光细胞化学染色结果证明SHISAL1-N抗体与细胞质中的SHISAL1蛋白特异性结合.结论成功优化SHISAL1抗原并制备了小鼠抗人SHISAL1多克隆抗体,可应用于Western blot实验、免疫沉淀和免疫荧光细胞化学染色.

Objective To investigate antigen optimization of Shisa like protein 1(SHISAL1)for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody.Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein,and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1,termed SHISAL1-N,was selected as the antigen.The coding region of SHISAL1-N was cloned by molecular cloning technique,and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid.The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21(DE3)bacteria and induced to express by IPTG.The two proteins were purified and immunized to female Kunming mice,respectively.The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis,immunoprecipitation and immunofluorescent cytochemical staining.Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed,and the two fused proteins,SHISAL1 and SHISAL1-N,were induced to express.Moreover,two types of SHISAL1 mouse polyclonal antibodies,derived from SHISAL1-N and SHISAL1 antigens,were obtained.Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein.Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm.Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully,which can be used for Western blot analysis,immunoprecipitation and immunofluorescence cytochemical staining.