摘要
目的 测定和分析人卵巢颗粒细胞中雌激素 β型受体 (ERβ)cDNA的核苷酸序列结构。方法 采用DMEM培养基、以PERCOLL方法从人体外受精卵泡液中制备颗粒细胞。根据TRIzolReagent试剂盒方法抽提RNA ,并在Dispocolumn柱中用寡聚 (dT)纯化mRNA。用SuperScriptTMⅡRT试剂盒反转录PCR合成cDNA。扩增产物与pGEM -T载体连接 ,并转化大肠杆菌XL1-Blue。测序按SequenaseVersion 2 .0DNA测序试剂盒提供的方法进行。核苷酸序列结构用DNAMAN软件包进行分析。结果 经两对引物扩增 ,获得长度为 14 95bp(其中包括 14 3 1bp阅读框 )的雌激素 β受体cDNA序列。根据阅读框编码的氨基酸序列 ,可分A/B、C、D、E/F功能区 ,其中C区富含半胱氨酸 ,为DNA结合区 (DBD) ;E/F区为配基结合区 (LBD)。结论 卵巢颗粒细胞中ERβ基因存在及序列分析结果证实了ERβ对卵巢颗粒细胞中雌激素自分泌的调节意义。
Objective ToanalyzethenucleotidesequenceofcDNAanddeducetheaminoacidsequence ofhumanestrogenreceptor (hERβ)inhumangranulosacells Methods Granulosacellswerepreparedfromthe ovaryofIVF ETcasesbyPercolltechniquewithDulbecco′smodifiedeaglemedium RNAwasextractedwiththe TRIzolreagentkit,andmRNAwaspurifiedwitholigo (dT) celluloseinasteriledispocolumn cDNAwas synthesizedbyRT PCRusingtheSuperScriptTMⅡRTkit AmplifiedproductswereclonedintothepGEM Tvector andtransfectedintoE coliXL1 Blue Thenucleotidesequencesweredeterminedbyrepeatedsequencingofboth strandsofalkaline denaturalplasmidDNAusingtheSequenaseVersion 2 0DNAsequencingkit Theobtained DNAsequencewascompiledandanalyzedusingDNAMANcomputerprograms Results AmplifiedcDNAof hERβinhumangranulosacellswascomposedof 14 95bp ,containinga 14 3 1bpopenreadingframe The predictedERβproteinconsistedof4 77aminoacids ThepredictedERproteinincluded 4functiondomains:A/B , C ,D ,andE/Fdomains Amongthesedomains,Cdomain ,richlycontainingcysteine ,wastheDNA binding domain (DBD) ,andE/Fdomainwastheligand bindingdomain (LBD) Conclusion DetectionofERβinthe ovarygranulosacellsplayedanimportantroleinexplainingtheself endocrinefunctionofestrogen
出处
《中华医学杂志》
CAS
CSCD
北大核心
2000年第1期28-30,共3页
National Medical Journal of China
