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目的构建带GST标签的人LC3B基因原核表达载体,得到GST-LC3B重组质粒并纯化出GST-LC3B融合蛋白,体外检测并证实该蛋白的生物学活性。方法利用PCR技术从人乳腺文库中扩增出LC3B基因的编码序列,将该序列插入到p GEX-KG载体中,得到GST-LC3B重组质粒,转化大肠杆菌Rossate,经小量诱导后利用GSTSepharose 4B亲和珠纯化GST-LC3B融合蛋白,通过SDS-PAGE电泳和Western印迹方法进行检测,GST pull-down技术证实其生物学活性。结果利用PCR技术从人乳腺文库中成功扩增得到大小约400 bp的目的基因片段,插入p GEX-KG载体中得到GST-LC3B质粒,经双酶切鉴定及测序结果表明重组质粒构建成功;转化Rossate菌并小量诱导,表达鉴定成功后纯化得到相对分子质量(Mr)约为40×103的目的蛋白;GST pull-down技术检测出GST-LC3B融合蛋白可以和Atg4B蛋白在体外作用,具有较好的生物学活性。结论成功构建了人自噬相关基因LC3B的原核表达产物,为进一步研究LC3B在自噬中的作用机制奠定了基础。
Objective To construct prokaryotic expression vector of human LC3B gene with GST tag, obtain GST-LC3B recombinant plasmid and purify GST-LC3B fusion protein, and detect and confirm the biological activity of this protein in vitro. Methods The coding sequence of LC3B gene was amplified from human mammary gland by PCR and inserted into pGEX-KG vector to obtain GST-LC3B recombinant plasmid. The recombinant plasmid was transformed into E. coli Rossate. After induced by GSTSepharose 4B The GST-LC3B fusion protein was purified by affinity beads and detected by SDS-PAGE electrophoresis and Western blotting. GST pull-down technique was used to confirm its biological activity. Results The gene fragment of about 400 bp was successfully amplified from the human breast cDNA library by PCR and inserted into pGEX-KG vector to obtain GST-LC3B plasmid. The double-stranded DNA was digested with restriction enzyme and sequencing results showed that the recombinant plasmid was successfully constructed. Rossate bacteria were induced in a small amount. After the expression was identified successfully, the target protein with the molecular weight (Mr) of about 40 × 103 was purified. GST pull-down assay showed that GST-LC3B fusion protein could interact with Atg4B protein in vitro, Good biological activity. Conclusion The prokaryotic expression product of human autophagy-related gene LC3B was successfully constructed, which laid the foundation for further study on the mechanism of LC3B in autophagy.