论文部分内容阅读
利用生物信息学方法分析乙酰氨基半乳糖转移酶14(GALNT14)蛋白序列,根据亲水性、抗原性、柔韧性及表面性等指标选择一段多肽序列作为抗原用于抗体制备。将该DNA片段插入pET-DsbA,构建原核表达载体pET-DsbA-GALNT14。用IPTG诱导表达蛋白,经镍柱纯化蛋白后免疫新西兰大白兔,获得GALNT14多克隆抗血清。用免疫扩散和免疫印迹检测抗体效价及特异性。结果显示,成功表达并纯化了目的蛋白。免疫动物后获得了效价较高、特异性强的GALNT14多克隆抗体,GALNT14蛋白在人乳腺癌和肾癌细胞株中均有表达。该结果为进一步研究GALNT14的功能及其在肿瘤发生发展的作用方面奠定了基础。
The bioinformatics method was used to analyze the protein sequence of GALNT14, and a polypeptide sequence was selected as the antigen for antibody preparation according to the indexes of hydrophilicity, antigenicity, flexibility and surface. The DNA fragment was inserted into pET-DsbA to construct prokaryotic expression vector pET-DsbA-GALNT14. The protein was induced with IPTG, and the protein was purified by nickel column and immunized New Zealand white rabbits to obtain polyclonal antiserum against GALNT14. Antibody titers and specificities were detected by immunodiffusion and immunoblotting. The results showed that the target protein was successfully expressed and purified. GALNT14 polyclonal antibody with high titer and high specificity was obtained after immunization of animals, and GALNT14 protein was expressed in human breast cancer and renal cell carcinoma cell lines. The results laid the foundation for further study on the function of GALNT14 and its role in tumorigenesis.