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目的体外构建HBsAg缺失型乙型肝炎病毒(HBV)复制细胞模型,并探讨HBsAg对HBV复制的影响。方法定点突变pch9质粒上HBV 1.1倍体基因S蛋白表达区,构建HBV1.1倍体HBsAg突变质粒,并进行测序鉴定;将鉴定正确的突变质粒瞬时转染HepG2细胞,设未突变pch9质粒转染细胞作为对照,采用ELISA法检测转染72 h的细胞培养上清中HBsAg的表达;Southern blot及Real-time PCR法检测转染72 h的细胞内HBV DNA的复制水平。结果定点突变质粒经测序证实构建正确;突变质粒转染72 h的细胞培养上清中HBsAg A450值为(0.06±0.003),表达呈阴性,对照细胞A450值为(1.82±0.010),表达呈阳性,转染突变质粒72 h的细胞内HBV DNA条带浓度明显高于转染未突变质粒的细胞;转染突变质粒的细胞内HBV DNA绝对拷贝数分别为9.33×106、5.26×106,约为转染未突变质粒(6.91×105)的7.6~13.5倍。结论已成功构建了HBsAg缺失型HBV复制细胞模型,为HBV DNA复制及相关研究提供了实验依据;推测HBsAg是HBV DNA形成的自限性因素,能够负调控HBVDNA复制。 相似文献
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1,4Cyclohexanedicarboxylicacid(1,4chdcH2)possessesachair typestructurewithcis andtrans conformations,soitcanconnectmetalionsindiffer entdirections.Hence,chdcmaybeagoodcandidate fortheconstructionofnovelcoordinationpoly mers[1-9].AsshowninFig.1[9],conformationAis Fig.1TransformsbetweenthedifferentconformationsofchdcH2theleaststableandisabletochangetoitsconformer Beasily.However,itisalittlemoredifficultwhenC becomesBbecausetheαprotonsshouldbedeproto natedtoacceleratetheequilibriumalthoughBi… 相似文献
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8-Hydroxyquinoline(HQ) and its derivatives areof the most intensivelyinvestigatedligands in the co-ordination chemistry[1 -3]. They have been used inmetallic ion detection,chromatography and especiallyin organic light-emitting diodes (OLEDS)[4]. Poly-oxometalates compounds have received muchattentionbecause of their potential applications in catalysis ,sorption, magnetism,photochemistry[5 ,6]. We origi-nally intended to gain the organic-inorganic hybridcomplex of HQ and polyoxometalates . … 相似文献
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目的构建瓣状内切核酸酶1(Flap endonuclease 1,FEN1)基因重组真核表达质粒,并进行鉴定。方法提取L02细胞总RNA,逆转录合成cDNA,经巢式PCR扩增FEN1基因,定向克隆至载体pcDNA3.1,构建重组真核表达质粒,经酶切及测序进行鉴定。将鉴定正确的真核表达质粒转染293T细胞,Western blot检测FEN1蛋白的表达。结果 FEN1基因重组真核表达质粒经酶切及测序鉴定证明构建正确;在相对分子质量约47 000处可见目的蛋白条带,转染细胞中FEN1蛋白表达量较空载体转染组及空白细胞对照组提高约3倍。结论已成功构建了FEN1基因重组真核表达质粒,并可在293T细胞中过表达FEN1。 相似文献
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