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目的:获得高质量及充足的Fas重组蛋白。方法:采用PCR构建表达重组质粒,亲和层析纯化Fas重组蛋白,ELISA检测其免疫学功能。结果:①PCR扩增获得目的片段约 470 bp。②Sanger双脱氧链终止法测序表明,该 DNA序列仅 174位由A→G突变,属于同义突变,与已知的Fas膜外区氨基酸完全一致。③重组质粒经IPTG诱导有一蛋白得以表达,其目的融合蛋白占细菌总蛋白的17. 4%,分子量约为 34．3 kD。④经 Factor Xa切割 Fas融合蛋白,纯化获得 Fas重组蛋白,Western印迹出现特异性Fas蛋白带,分子量为21．3kD,与预测结果相吻合。⑤ELISA检测 Fas重组蛋白,具有抗体的免疫学活性。结论:成功地表达并纯化了Fas重组蛋白,解决了Fas不易获得的难题,为研究Fas提供了良好的实验材料。
Objective: To obtain high quality and sufficient Fas recombinant protein. Methods: The recombinant plasmids were constructed by PCR, and the recombinant Fas protein was purified by affinity chromatography. The immunological function was detected by ELISA. Results: ① The target fragment was about 470 bp amplified by PCR. ②Sanger dideoxy chain termination sequencing showed that only 174 of the DNA sequence was mutated from A → G, which were synonymous mutations, which were completely consistent with the known extracellular amino acids of Fas membrane. The recombinant plasmid induced by IPTG to express a protein, the purpose of the fusion protein accounted for 17.4% of total bacterial protein, the molecular weight of about 34.3 kD. ④ The Fas fusion protein was cut by Factor Xa, and the recombinant Fas protein was obtained by purification. The specific Fas protein band was detected by Western blotting and its molecular weight was 21.3 kD, which was consistent with the predicted results. ⑤ ELISA detection of Fas recombinant protein, with the antibody immunological activity. Conclusion: The recombinant Fas protein was successfully expressed and purified, which solved the problem that Fas was not easy to obtain and provided a good experimental material for the study of Fas.