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1、第30卷第1期第三軍醫(yī)大學(xué)學(xué)報Vol.30,No.12008年1月ACTAACADEMIAEMEDICINAEMILITARISTERTIAEJan.200819文章編號:100025404(2008)0120019204論著人重組蛋白P311的原核表達(dá)純化與鑒定譚江琳,袁順宗,彭旭,馬兵,羅高興,吳軍(第三軍醫(yī)大學(xué)西南醫(yī)院全軍燒傷研究所,疾病蛋白質(zhì)組學(xué)重慶市市級重點實驗室,創(chuàng)傷,燒傷與復(fù)合傷國家重點實驗室,重慶400038)摘要:目的原核表達(dá)人增生性瘢痕新候選相關(guān)蛋白P311,并對該重組蛋白進(jìn)行純化、鑒定。方法通過PCR方法擴(kuò)增人P311基因,利用BamHⅠ和XhoⅠ酶切
2、位點將其克隆至谷胱甘肽2S2轉(zhuǎn)移酶(glutathione2s2transferase,GST)融合蛋白表達(dá)載體pGEX24T21中,轉(zhuǎn)化大腸桿菌BL21(DE3)后,經(jīng)異丙基硫代2β2D2半乳糖苷(isopropyl2β2D252thiogalactoside,IPTG)誘導(dǎo)表達(dá),用SDS2PAGE、Westernblotting等方法鑒定表達(dá)產(chǎn)物,并用谷胱甘肽2瓊脂糖小珠親和純化表達(dá)的GST2P311蛋白。結(jié)果重組載體pGEX24T2P311經(jīng)酶切與測序鑒定證實構(gòu)建成功。導(dǎo)入大腸桿菌進(jìn)行表達(dá),表達(dá)產(chǎn)物相對分子量為34KD左右,與預(yù)期值相符。該條帶經(jīng)免疫印跡檢測鑒定為GS
3、T抗體陽性,獲得了純化的GST2P311蛋白。結(jié)論構(gòu)建了pGEX24T2P311原核表達(dá)載體,并在大腸桿菌BL21(DE3)中成功表達(dá),親和純化獲得較高純度的GST融合蛋白,為下一步繼續(xù)研究P311的生物學(xué)功能奠定了基礎(chǔ)。關(guān)鍵詞:P311;原核表達(dá);增生性瘢痕中圖法分類號:R341;R394233文獻(xiàn)標(biāo)識碼:AProkaryoticexpression,purificationandidentificationofhumanP311proteinTANJiang2lin,YUANShun2zong,PENGXu,MABing,LUOGao2xing,WUJun(StateK
4、eyLaboratoryofTrauma,BurnsandCombinedInjury,InstituteofBurns,SouthwestHospital;ChongqingKeyLaboratoryofProteomicsofDiseases,Chongqing400038,China)Abstract:ObjectiveToexpress,purifyandidentifyhumanP311protein,acandidateproteininhyper2trophicscarformation.MethodsThecDNAsequenceofhumanP311obt
5、ainedbyPCRwasclonedintotheprokaryoticexpressionvectorpGEX24T21containingglutathione2s2transferase(GST).pGEX24T2P311wastransformedintoE.coliBL21(DE3).TheexpressionofGST2P311fusionproteinwasinducedbyIPTG,identi2fiedbybothSDS2PAGEandWesternblotting,andthenpurifiedwithglutathione2sepharosebead
6、s.ResultsTherestrictionendonucleasedigestionandthesequencingofrecombinantplasmiddemonstratedthatpGEX24T23P311vectorwassuccessfullyconstructed.Therelativemolecularweightofexpressedproteinwasabout34×10whichwasasthesameastheobjectiveprotein,andthecorrespondingproteinwasidentifiedbytheimmunobl
7、ot2tingwithantibodyagainstGST.ConclusionpGEX24T2P311vectoriscorrectlyconstructed,andtheGST2P311fusionproteinissuccessfullyexpressedinE.coliBL21(DE3)andpurified.Keywords:P311;prokaryoticexpression;hypertrophicscar增生性瘢痕(hypertrophicscar,HS)是皮膚創(chuàng)傷異既往研究表明成纖維細(xì)胞向肌成纖維
