云南阿昌族G6PD基因突变G487A在DF213中的表达
운남아창족G6PD기인돌변G487A재DF213중적표체
EXPRESSION OF G6PD GENE G487A MUTATION IN DF213 FROM ACHANG PEOPLE OF YUNNAN
  • 万方数据
    生物物理学报 생물물이학보
    ACTA BIOPHYSICA SINICA
    2007年 1期 20-28 ,共9页

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    양은봉%주월춘%리홍균%리치강%려회여%리단이%최영파%풍유양%여과우%황우광

    阿昌族%葡萄糖-6-磷酸脱氢酶%基因%表达载体 阿昌族%葡萄糖-6-燐痠脫氫酶%基因%錶達載體
    아창족%포도당-6-린산탈경매%기인%표체재체
    为获得阿昌族G6PDWT和G6PDG487A重组蛋白,研究G6PDG487A的结构和功能改变,从云南省德宏州梁河县杞木寨乡湾中村阿昌族聚集地的G6PD缺陷家系先证者和正常阿昌族个体全血提取RNA,经RT-巢式PCR得cDNA,将cDNA克隆至pMD18-T simple载体中并测序;错配碱基经定点突变修复后,目的基因亚克隆至pThioHis(A)载体,构建了阿昌族G6PD基因野生型和G487A突变型原核表达载体:pThioHis(A)-AChang-G6PDWT和pThioHis(A)-AChang-G6PDG487A.用重组质粒转化E.coli Competent Cells DF213(G6PD defeciency),经IPTG诱导G6PD表达、10%SDS-PAGE电泳检测表达蛋白和紫外340 nm定量测定G6PD活性的分析表明,pThioHis(A)-AChang-G6PDWr和pThioHis(A)-AChang-G6PDG487A在DF213中成功表达,分子量约为59 kDa.IPTG诱导0、3、6、9、和12 h后,G6PD活性逐渐增高,G6PD基因WT表达的酶活性约是G487A的20～25倍.表达载体的构建以及G6PD cDNA在DF213中成功表达,为重组酶G6PDG487A的进一步研究奠定了基础.
    위획득아창족G6PDWT화G6PDG487A중조단백,연구G6PDG487A적결구화공능개변,종운남성덕굉주량하현기목채향만중촌아창족취집지적G6PD결함가계선증자화정상아창족개체전혈제취RNA,경RT-소식PCR득cDNA,장cDNA극륭지pMD18-T simple재체중병측서;착배감기경정점돌변수복후,목적기인아극륭지pThioHis(A)재체,구건료아창족G6PD기인야생형화G487A돌변형원핵표체재체:pThioHis(A)-AChang-G6PDWT화pThioHis(A)-AChang-G6PDG487A.용중조질립전화E.coli Competent Cells DF213(G6PD defeciency),경IPTG유도G6PD표체、10%SDS-PAGE전영검측표체단백화자외340 nm정량측정G6PD활성적분석표명,pThioHis(A)-AChang-G6PDWr화pThioHis(A)-AChang-G6PDG487A재DF213중성공표체,분자량약위59 kDa.IPTG유도0、3、6、9、화12 h후,G6PD활성축점증고,G6PD기인WT표체적매활성약시G487A적20～25배.표체재체적구건이급G6PD cDNA재DF213중성공표체,위중조매G6PDG487A적진일보연구전정료기출.
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