论文部分内容阅读
目的获得原核表达的OY-TES-1氨基端截短蛋白(OY-TES-1-N),并制备其多克隆抗体。方法扩增编码OY-TES-1-N 268个氨基酸(A6-R273)的cDNA序列;将PCR产物插入原核表达载体pMAL-C2,构建重组质粒,并转化DH5α菌;通过蓝白斑筛选、DNA测序筛出阳性菌;在优化条件下用IPTG对阳性菌进行诱导表达MBP/OY-TES-1-N融合蛋白;上Amyloseresin亲和层析柱纯化,行Western blot鉴定。以融合蛋白作为抗原免疫新西兰白兔制备抗血清,经活化琼脂糖微球纯化后,采用ELISA法及Western blot法分别检测抗血清的效价和多克隆抗体的特异性。结果成功诱导表达出MBP/OY-TES-1-N融合蛋白。以该蛋白免疫新西兰兔制备抗血清,抗体效价为1∶1 000,Western blot检测证实该抗体能与目的蛋白发生特异性结合。结论成功地表达并纯化了MBP/OY-TES-1-N融合蛋白,并制备了特异性多克隆抗体。
Objective To obtain OY-TES-1 amino-terminal truncated protein (OY-TES-1-N) and to prepare its polyclonal antibody. Methods The cDNA sequence encoding 268 amino acids (A6-R273) of OY-TES-1-N was amplified by PCR. The PCR product was inserted into prokaryotic expression vector pMAL-C2 to construct recombinant plasmid and transformed into DH5α. The positive bacteria were screened out and the positive bacteria were induced to express MBP / OY-TES-1-N fusion protein by IPTG under the optimal conditions. The recombinant protein was purified by Amyloseresin affinity chromatography and identified by Western blot. The fusion protein was used as an antigen to immunize New Zealand white rabbits to prepare antiserum. After purified by activated agarose microspheres, the antiserum titer and the specificity of polyclonal antibody were detected by ELISA and Western blot respectively. Results The MBP / OY-TES-1-N fusion protein was successfully induced. The protein was used to immunize New Zealand rabbits to prepare antiserum. The titer of antibody was 1: 1 000. Western blot showed that the antibody could specifically bind to the target protein. Conclusion The MBP / OY-TES-1-N fusion protein was successfully expressed and purified, and specific polyclonal antibodies were prepared.