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1、第30卷第1期第三军医大学学报Vol.30,No.12008年1月ACTAACADEMIAEMEDICINAEMILITARISTERTIAEJan.200819文章编号:100025404(2008)0120019204论著人重组蛋白P311的原核表达纯化与鉴定谭江琳,袁顺宗,彭旭,马兵,罗高兴,吴军(第三军医大学西南医院全军烧伤研究所,疾病蛋白质组学重庆市市级重点实验室,创伤,烧伤与复合伤国家重点实验室,重庆400038)摘要:目的原核表达人增生性瘢痕新候选相关蛋白P311,并对该重组蛋白进行纯化、鉴定。方法通过PCR方法扩增人P311基因,利用BamHⅠ和XhoⅠ酶切
2、位点将其克隆至谷胱甘肽2S2转移酶(glutathione2s2transferase,GST)融合蛋白表达载体pGEX24T21中,转化大肠杆菌BL21(DE3)后,经异丙基硫代2β2D2半乳糖苷(isopropyl2β2D252thiogalactoside,IPTG)诱导表达,用SDS2PAGE、Westernblotting等方法鉴定表达产物,并用谷胱甘肽2琼脂糖小珠亲和纯化表达的GST2P311蛋白。结果重组载体pGEX24T2P311经酶切与测序鉴定证实构建成功。导入大肠杆菌进行表达,表达产物相对分子量为34KD左右,与预期值相符。该条带经免疫印迹检测鉴定为GS
3、T抗体阳性,获得了纯化的GST2P311蛋白。结论构建了pGEX24T2P311原核表达载体,并在大肠杆菌BL21(DE3)中成功表达,亲和纯化获得较高纯度的GST融合蛋白,为下一步继续研究P311的生物学功能奠定了基础。关键词:P311;原核表达;增生性瘢痕中图法分类号:R341;R394233文献标识码: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)是皮肤创伤异既往研究表明成纤维细胞向肌成纤维
