中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
11期
967-971
,共5页
郑筱娇%高洲%沈蓉蓉%赵行%岑东%罗建平%吕建新%裴仁治%滑世轩
鄭篠嬌%高洲%瀋蓉蓉%趙行%岑東%囉建平%呂建新%裴仁治%滑世軒
정소교%고주%침용용%조행%잠동%라건평%려건신%배인치%활세헌
肝细胞生长因子%蛋白%重组%纯化%活性
肝細胞生長因子%蛋白%重組%純化%活性
간세포생장인자%단백%중조%순화%활성
Hepatocyte growth factor%Protein%Recombinant%Purification%Activity
目的 通过构建肝细胞生长因子(HGF)基因的重组原核表达载体,转化大肠杆菌,制备HGF重组蛋白并初步证实其活性.方法 克隆HGF基因插入载体pET-26b(+),构建重组原核表达载体pET-26b(+)-HGF,转化E.coli Rosseta(DE3).IPTG诱导转化菌表达重组蛋白,采用Ni-NTA树脂亲和层析法进行纯化,复性后冷冻干燥.结果 HGF基因重组原核表达载体pET-26b(+)-HGF构建成功.转化pET-26b(+)-HGF的E.coli Rosseta(DE3)以包涵体形式大量表达目的蛋白,占菌体总蛋白的38％,并经Western blot证实.经Ni-NTA树脂亲和层析法纯化后HGF蛋白纯度约为95％,作用于人非小细胞肺癌细胞系A549细胞发现可促进其增殖、迁徙并抑制凋亡.结论 成功构建了HGF基因重组原核表达载体pET-26b(+)-HGF,转化E.coli Rosseta(DE3)后成功表达,纯化复性回复重组HGF蛋白结构,经体外实验证实具有生物学活性.
目的 通過構建肝細胞生長因子(HGF)基因的重組原覈錶達載體,轉化大腸桿菌,製備HGF重組蛋白併初步證實其活性.方法 剋隆HGF基因插入載體pET-26b(+),構建重組原覈錶達載體pET-26b(+)-HGF,轉化E.coli Rosseta(DE3).IPTG誘導轉化菌錶達重組蛋白,採用Ni-NTA樹脂親和層析法進行純化,複性後冷凍榦燥.結果 HGF基因重組原覈錶達載體pET-26b(+)-HGF構建成功.轉化pET-26b(+)-HGF的E.coli Rosseta(DE3)以包涵體形式大量錶達目的蛋白,佔菌體總蛋白的38％,併經Western blot證實.經Ni-NTA樹脂親和層析法純化後HGF蛋白純度約為95％,作用于人非小細胞肺癌細胞繫A549細胞髮現可促進其增殖、遷徙併抑製凋亡.結論 成功構建瞭HGF基因重組原覈錶達載體pET-26b(+)-HGF,轉化E.coli Rosseta(DE3)後成功錶達,純化複性迴複重組HGF蛋白結構,經體外實驗證實具有生物學活性.
목적 통과구건간세포생장인자(HGF)기인적중조원핵표체재체,전화대장간균,제비HGF중조단백병초보증실기활성.방법 극륭HGF기인삽입재체pET-26b(+),구건중조원핵표체재체pET-26b(+)-HGF,전화E.coli Rosseta(DE3).IPTG유도전화균표체중조단백,채용Ni-NTA수지친화층석법진행순화,복성후냉동간조.결과 HGF기인중조원핵표체재체pET-26b(+)-HGF구건성공.전화pET-26b(+)-HGF적E.coli Rosseta(DE3)이포함체형식대량표체목적단백,점균체총단백적38％,병경Western blot증실.경Ni-NTA수지친화층석법순화후HGF단백순도약위95％,작용우인비소세포폐암세포계A549세포발현가촉진기증식、천사병억제조망.결론 성공구건료HGF기인중조원핵표체재체pET-26b(+)-HGF,전화E.coli Rosseta(DE3)후성공표체,순화복성회복중조HGF단백결구,경체외실험증실구유생물학활성.
Objective To prepare hepatocyte growth factor(HGF) recombinant protein and confirm its activity preliminarily according to building HGF gene prokaryotic expression vector and transforming into E.coli.Methods Clone HGF inserted into the vector pET-26b(+) to construct prokaryotic expression vector pET-26b(+)-HGF and transform into E.coli Rosseta(DE3).The transformed bacteria induced by IPTG was purified through Ni-NTA resin affinity chromatography frozen-drying after renaturation.Results HGF gene recombinant prokaryotic expression vector pET-26b(+)-HGF was constructed successfully.E.coli Rosseta(DE3) which was transformed into pET-26b(+)-HGF expresses the target protein as the form of inclusion bodies,accounting for 38％ of the total bacterial proteins,and confirmed by Western blot.HGF protein which was purified by Ni-NTA resin affinity chromatography,has a purity of about 95％,and can promote proliferation,migration,and inhibition of apoptosis for human non-small cell lung cancer cell line A549 cells after interaction.Conclusion HGF gene recombinant prokaryotic expression vector pET-26b (+)-HGF was constructed and expressed in transformed E.coli Rosseta(DE3) successfully.They resumed their recombinant HGF protein structure after purification and renaturation,and had biological activity confirmed by in vitro studies.
