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1、第31卷第2期暨南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)VO1.31No.22010年4月JounlalofJinanUniversity(MedicineEdition)Apr.2010小鼠附睪特異性輔脂酶的原核表達(dá)及其抗體的制備關(guān)藝青,禹艷紅,黃丹,潘善培(暨南大學(xué)生命科學(xué)技術(shù)學(xué)院生殖免疫研究所,廣東廣州510632)[摘要]目的:利用原核表達(dá)系統(tǒng)重組表達(dá)小鼠附睪特異性輔脂酶(meClps),并制備抗meClps抗體。方法:通過(guò)生物信息分析meClps蛋白結(jié)構(gòu),Oligo6設(shè)計(jì)引物,PCR法從小鼠附睪eDNA中擴(kuò)增胱C全長(zhǎng)序列并克隆到pMD19一T載體中。將含腳c序列pMD19一T載體通過(guò)PC
2、R擴(kuò)增成熟肽區(qū)段序列,重組到pET-21b原核表達(dá)載體上,經(jīng)菌落PCR及測(cè)序鑒定后,轉(zhuǎn)化到表達(dá)菌株EcoliBL21(DE3),經(jīng)IPTG誘導(dǎo)蛋白表達(dá)后用Trieine.SDS.PAGE和Westernblotting檢測(cè)重組meClps的表達(dá)。包涵體沉淀經(jīng)濃度為2mol/L尿素洗滌,Trieine.SDS.PAGE分離、染色、脫色,切下目的條帶用生理鹽水浸泡、磨碎與弗氏佐劑混合后免疫新西蘭大白兔獲取免疫血清,Westernblotting檢測(cè)抗體與meClps反應(yīng)。結(jié)果:在原核表達(dá)系統(tǒng)成功重組表達(dá)meClps,目的蛋白主要以包涵體形式存在,制備了高效價(jià)的兔抗meClps抗體。
3、結(jié)論:成功建立meClps的原核表達(dá)系統(tǒng)和獲得兔抗meClps抗體。[關(guān)鍵詞]附睪;輔脂酶;原核表達(dá);抗體[中圖分類號(hào)】R715.2[文獻(xiàn)標(biāo)志碼】A[文章編號(hào)】1000—9965(2010)02—0105—06Prokaryoticexpressionofmouseepididymis-specificcolipaseandpreparationofitsantibodyGUANYi—qing,YUYan—hong,HUANGDan,PANShan—pei(InstituteofReproductiveImmunology,ColegeofLifeScienceandTechn
4、ology,JinanUniversity,Guangzhou510632,P.R.China)[Abstract]Ainl:Toexpressmouseepididymis—specificcolipase(meClps)inpmkaryoticsystemandpreparetheanti—meClpsantibody.Methods:ThemeClpsproteinstructurewasanalysedbyusingbioin—formation,andprimersweredesignedwithOligo6.Thefull—lengthofmeClpswasampl
5、ifiedfrommouseepididymalcDNAandclonedintopMD]9一Tvector.ThesequenceofmaturepeptidewasobtainedbyPCRfrompMD19一TvectorwithmeClps.Thentheproductwasrecombinedintothepmkaryoticexpressionvec—torpET-21b.TherecombinantplasmidwasidentifiedbyclonePCRandsequencingbefore~ansformationintocoliBL21(DE3).Afte
6、rinducedbyIPTG,therecombinantmeClpsproteinwasexpressedandsepreatedbyTrieine—SDS—PAGE,andthenidentifiedbyWesternblotting.Theinclusionbodieswerewashedwith2mol/LureaandsepreatedbyTricine—SDS—PAGE.Afterstaininganddestaining,thegelcontainingmeClpsproteinwascut,equilibratedwithphysiologicalsalines
7、olution,rubbed,andemulsi—fledwithFreundadjuvant,thenusedasantigentoimmunizerabbitandprepareantibodies.Theanti-meClpsantibodyresponsewasidentifiedbyWesternblotting.Results:ThemeClpsproteinwassuccess-fullyexpressedinprokaryoticsystem.butmainlyininclu