目的 研究Shisa类蛋白1（SHISAL1）的抗原优化，制备小鼠抗人SHISAL1多克隆抗体，并鉴定所制备抗体的特异性。 方法 应用生物信息学预测SHISAL1蛋白的抗原表位区域，然后选择由28～97位氨基酸残基组成的多肽，命名为SHISAL1-N，作为抗原。通过分子克隆技术克隆SHISAL1-N编码区，然后将其插入pET-28a中，构建pET28a-SHISAL1-N重组质粒。将两个重组质粒pET28a-SHISAL1-N和pET28a-SHISAL1转化到BL21（DE3）菌中，用IPTG诱导表达。分别纯化这两种蛋白，用于免疫雌性昆明小鼠。通过Western blot分析、免疫共沉淀和免疫荧光细胞化学染色来检测所获取抗体的特异性和灵敏度。 结果 成功构建pET28a-SHISAL1-N重组质粒，并诱导表达两种融合蛋白，即SHISAL1和SHISAL1-N。此外，获得了两种来源于SHISAL1-N和SHISAL1抗原的SHISAL1小鼠多克隆抗体。Western blot结果显示，与由SHISAL1抗原制备的抗体相比，由SHISAL1-N抗原制备的抗体特异性和灵敏度更高，能够特异性识别不同内源性SHISAL1蛋白。免疫共沉淀结果表明，SHISAL1-N抗体能够特异性地沉淀肝细胞癌细胞中的SHISAL1蛋白，免疫荧光结果表明，SHISAL1-N抗体能够特异性地结合细胞质中的SHISAL1蛋白。 结论 我们成功地优化了SHISAL1抗原并制备了小鼠抗人SHISAL1多克隆抗体，可以用于Western blot分析、免疫共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.