人自噬相关基因LC3B原核表达载体的构建及活性检测

Construction of prokaryotic expression vector with human autophagy-related LC3B gene and its activity detection

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 the prokaryotic expression vector of human autophagy-related LC3B gene, obtain the GST-LC3B recombinant plasmid, purify the GST-LC3B fusion protein and identify its activity in vitro. Methods Human LC3B coding region was amplified from the human mammary gland cDNA by PCR and inserted into the prokaryotic expres- sion vector pGEX-KG. The recombinant plasmid pGEX-KG-LC3B was transformed into E. coil Rossate. The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis. The function of the purified protein GST-LC3B was detected by GST pull-down assay. Results About 400 bp of the LC3B coding region was successfully amplified from the mammary gland library by PCR and inserted into pGEX-KG. The result of double digestion and sequencing showed that the GST-LC3B recombinant plasmid was successfully obtained. The GST-LC3B fusion pro- tein of about 40 000 （Mr） was successfully purified and identified by SDS-PAGE and Western blotting analysis. GST pull- down assay showed that GST-LC3B could interact with Atg4B, which identified its known function. Conclusion The pro- karyotic expression vector of GST-LC3B is constructed successfully, which will facilitate further research on the function of LC3B in autophagy.