军事医学
軍事醫學
군사의학
BULLETIN OF THE ACADEMY OF MILITARY MEDICAL SCIENCES
2014年
11期
867-870
,共4页
黄蓉%徐小洁%梁迎春%王涛%冯滢滢%周丽英%李玲%冀全博%郭靖%叶棋浓%杜楠
黃蓉%徐小潔%樑迎春%王濤%馮瀅瀅%週麗英%李玲%冀全博%郭靖%葉棋濃%杜楠
황용%서소길%량영춘%왕도%풍형형%주려영%리령%기전박%곽정%협기농%두남
人LC3B基因%原核表达%纯化%自噬
人LC3B基因%原覈錶達%純化%自噬
인LC3B기인%원핵표체%순화%자서
human LC3B%prokaryotic expression%purification%autophagy
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pGEX-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GST-Sepharose 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pGEX-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LC3 B在自噬中的作用机制奠定了基础。
目的：構建帶GST標籤的人LC3B基因原覈錶達載體，得到GST-LC3B重組質粒併純化齣GST-LC3B融閤蛋白，體外檢測併證實該蛋白的生物學活性。方法利用PCR技術從人乳腺文庫中擴增齣LC3B基因的編碼序列，將該序列插入到pGEX-KG載體中，得到GST-LC3B重組質粒，轉化大腸桿菌Rossate，經小量誘導後利用GST-Sepharose 4B親和珠純化GST-LC3B融閤蛋白，通過SDS-PAGE電泳和Western印跡方法進行檢測，GST pull-down技術證實其生物學活性。結果利用PCR技術從人乳腺文庫中成功擴增得到大小約400 bp的目的基因片段，插入pGEX-KG載體中得到GST-LC3B質粒，經雙酶切鑒定及測序結果錶明重組質粒構建成功；轉化Rossate菌併小量誘導，錶達鑒定成功後純化得到相對分子質量（ Mr）約為40×103的目的蛋白；GST pull-down技術檢測齣GST-LC3B融閤蛋白可以和Atg4B蛋白在體外作用，具有較好的生物學活性。結論成功構建瞭人自噬相關基因LC3B的原覈錶達產物，為進一步研究LC3 B在自噬中的作用機製奠定瞭基礎。
목적：구건대GST표첨적인LC3B기인원핵표체재체，득도GST-LC3B중조질립병순화출GST-LC3B융합단백，체외검측병증실해단백적생물학활성。방법이용PCR기술종인유선문고중확증출LC3B기인적편마서렬，장해서렬삽입도pGEX-KG재체중，득도GST-LC3B중조질립，전화대장간균Rossate，경소량유도후이용GST-Sepharose 4B친화주순화GST-LC3B융합단백，통과SDS-PAGE전영화Western인적방법진행검측，GST pull-down기술증실기생물학활성。결과이용PCR기술종인유선문고중성공확증득도대소약400 bp적목적기인편단，삽입pGEX-KG재체중득도GST-LC3B질립，경쌍매절감정급측서결과표명중조질립구건성공；전화Rossate균병소량유도，표체감정성공후순화득도상대분자질량（ Mr）약위40×103적목적단백；GST pull-down기술검측출GST-LC3B융합단백가이화Atg4B단백재체외작용，구유교호적생물학활성。결론성공구건료인자서상관기인LC3B적원핵표체산물，위진일보연구LC3 B재자서중적작용궤제전정료기출。
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.coli 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 diges-tion 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.
