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目的构建人类磷脂酰肌醇蛋白聚糖3(Glypican3,GPC3)重组原核表达载体。方法以人类肝脏cDNA文库为模板扩增出GPC3基因产物,与pMD-18 simple T载体进行连接构建出T克隆载体;从T载体上获取基因片段,并对pET-32a(+)空载体进行双酶切,用T4连接酶连接,构建出pET-32a(+)-GPC3原核表达载体;对构建好的载体再次测序,并进行诱导表达。结果 PCR产物长度为837 bp,即扩增的GPC3 C末端长度,与NCBI网站的相比较,序列完全一致,说明pET-32a(+)载体中正确插入了目的片段,并且诱导表达出蛋白。结论成功构建了融合表达Pet-GPC3载体,蛋白诱导表达成功。
Objective To construct human prokaryotic expression vector Glypican3 (GPC3). Methods The human liver cDNA library was used as a template to amplify the GPC3 gene product and ligated with the pMD-18 simple T vector to construct the T cloning vector. The gene fragment was obtained from the T vector and the pET-32a (+ Digested with T4 ligase and ligated with T4 ligase to construct the prokaryotic expression vector pET-32a (+) - GPC3. The constructed vector was sequenced again and induced to express. Results The length of PCR product was 837 bp, which was the length of the amplified GPC3 C terminal. Compared with the NCBI website, the sequence of the PCR product was exactly the same, indicating that the target fragment was correctly inserted into the pET-32a (+) vector and the protein was induced. Conclusion The fusion protein Pet-GPC3 was successfully constructed and the protein was induced to express successfully.