目的 :探讨人类泛素偶联酶E2C(ubiquitin-conjugating enzyme E2C,UBE2C)基因沉默对去势抵抗性前列腺癌PC3细胞增殖和侵袭的影响。方法:应用RT-PCR及蛋白质印迹法检测前列腺癌LNCap、PC3、DU145和C4-2细胞中UBE2C m RNA及蛋白的表达水...目的 :探讨人类泛素偶联酶E2C(ubiquitin-conjugating enzyme E2C,UBE2C)基因沉默对去势抵抗性前列腺癌PC3细胞增殖和侵袭的影响。方法:应用RT-PCR及蛋白质印迹法检测前列腺癌LNCap、PC3、DU145和C4-2细胞中UBE2C m RNA及蛋白的表达水平。将UBE2C si RNA转染入PC3细胞后,应用RT-PCR及蛋白质印迹法检测UBE2C m RNA及蛋白的表达水平,MTT法检测细胞的增殖能力,Transwell侵袭实验检测细胞的侵袭能力,蛋白质印迹法检测细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)、磷酸化ERK1/2(phospho-ERK1/2,p-ERK1/2)、蛋白激酶B(protein kinase B,PKB,又称Akt)和磷酸化Akt(phospho-Akt,p-Akt)蛋白的表达水平。结果:PC3、DU145和C4-2细胞中UBE2C m RNA和蛋白的表达水平显著高于LNCap细胞(P值均<0.05)。UBE2C si RNA转染后,PC3细胞中UBE2C m RNA及蛋白的表达水平均被抑制(P值均<0.05);UBE2C si RNA转染组PC3细胞的增殖能力和侵袭能力显著下降(P值均<0.01),p-ERK1/2和p-Akt蛋白的表达水平被显著抑制(P值均<0.01)。结论 :UBE2C基因沉默能够显著抑制去势抵抗性前列腺癌PC3细胞的增殖和侵袭,这一作用可能与下调ERK信号通路蛋白的活化有关。展开更多
A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which ...A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which encoded an abundance of known B-cell epitopes of the protein.The amplified fragment was inserted into pET-30a plasmid(Novagen)via two unique endonuclease restriction sites,Nco I and Sal I.Sequencing confirmed that the open reading frame of interest was correctly inserted into the positive recombinant plasmid.The positive plasmid was transformed into the host bacteria BL21(DE3)pLys for protein expression.After induction by IPTG at 37℃for 5 hours,the expressed product was analyzed by SDSPAGE and Western blotting,confirming successful expression.The product is a 23kDa fusion protein and was shown to react with sera derived from FMDV-infected animals.This approach provides an useful antigen for establishing an enzyme-linked immunoelectro-transfer blot assay(EITB)diagnostic method,useful for differentiating FMDV-infected animals from those that been vaccinated.展开更多
目的：为了探讨Rap2c基因与肺癌发生的关系，克隆人Ras家族小G蛋白Rap2c的cDNA，构建其真核表达质粒并在人肺癌细胞株A549和H1299中表达。方法人骨肉瘤细胞株U2OS提取细胞总RNA，经逆转录聚合酶链反应（RT-PCR）逆转录成cDNA，PCR扩增Ra...目的：为了探讨Rap2c基因与肺癌发生的关系，克隆人Ras家族小G蛋白Rap2c的cDNA，构建其真核表达质粒并在人肺癌细胞株A549和H1299中表达。方法人骨肉瘤细胞株U2OS提取细胞总RNA，经逆转录聚合酶链反应（RT-PCR）逆转录成cDNA，PCR扩增Rap2c，酶切后插入pcDNA3．1（＋）构建真核表达质粒 pcD-NA3．1（＋）-Rap2c，采用酶切及测序鉴定。重组质粒转染A 549和H 1299细胞，Western blot 检测其目的基因表达。结果双酶切及测序结果显示重组质粒pcDNA3．1（＋）-Rap2c成功构建，Werstern blot 检测到 A549和H 1299细胞有相应蛋白表达。结论成功构建人Rap2c基因真核表达质粒，为后续研究奠定了基础。展开更多
文摘目的 :探讨人类泛素偶联酶E2C(ubiquitin-conjugating enzyme E2C,UBE2C)基因沉默对去势抵抗性前列腺癌PC3细胞增殖和侵袭的影响。方法:应用RT-PCR及蛋白质印迹法检测前列腺癌LNCap、PC3、DU145和C4-2细胞中UBE2C m RNA及蛋白的表达水平。将UBE2C si RNA转染入PC3细胞后,应用RT-PCR及蛋白质印迹法检测UBE2C m RNA及蛋白的表达水平,MTT法检测细胞的增殖能力,Transwell侵袭实验检测细胞的侵袭能力,蛋白质印迹法检测细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)、磷酸化ERK1/2(phospho-ERK1/2,p-ERK1/2)、蛋白激酶B(protein kinase B,PKB,又称Akt)和磷酸化Akt(phospho-Akt,p-Akt)蛋白的表达水平。结果:PC3、DU145和C4-2细胞中UBE2C m RNA和蛋白的表达水平显著高于LNCap细胞(P值均<0.05)。UBE2C si RNA转染后,PC3细胞中UBE2C m RNA及蛋白的表达水平均被抑制(P值均<0.05);UBE2C si RNA转染组PC3细胞的增殖能力和侵袭能力显著下降(P值均<0.01),p-ERK1/2和p-Akt蛋白的表达水平被显著抑制(P值均<0.01)。结论 :UBE2C基因沉默能够显著抑制去势抵抗性前列腺癌PC3细胞的增殖和侵袭,这一作用可能与下调ERK信号通路蛋白的活化有关。
基金supported by the National Key Technolo-gies R&D Program(2006BAD06A12)the Key Project of Chinese National Programs for Fundamental Research and Development(2005CB523201)
文摘A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which encoded an abundance of known B-cell epitopes of the protein.The amplified fragment was inserted into pET-30a plasmid(Novagen)via two unique endonuclease restriction sites,Nco I and Sal I.Sequencing confirmed that the open reading frame of interest was correctly inserted into the positive recombinant plasmid.The positive plasmid was transformed into the host bacteria BL21(DE3)pLys for protein expression.After induction by IPTG at 37℃for 5 hours,the expressed product was analyzed by SDSPAGE and Western blotting,confirming successful expression.The product is a 23kDa fusion protein and was shown to react with sera derived from FMDV-infected animals.This approach provides an useful antigen for establishing an enzyme-linked immunoelectro-transfer blot assay(EITB)diagnostic method,useful for differentiating FMDV-infected animals from those that been vaccinated.
文摘目的：为了探讨Rap2c基因与肺癌发生的关系，克隆人Ras家族小G蛋白Rap2c的cDNA，构建其真核表达质粒并在人肺癌细胞株A549和H1299中表达。方法人骨肉瘤细胞株U2OS提取细胞总RNA，经逆转录聚合酶链反应（RT-PCR）逆转录成cDNA，PCR扩增Rap2c，酶切后插入pcDNA3．1（＋）构建真核表达质粒 pcD-NA3．1（＋）-Rap2c，采用酶切及测序鉴定。重组质粒转染A 549和H 1299细胞，Western blot 检测其目的基因表达。结果双酶切及测序结果显示重组质粒pcDNA3．1（＋）-Rap2c成功构建，Werstern blot 检测到 A549和H 1299细胞有相应蛋白表达。结论成功构建人Rap2c基因真核表达质粒，为后续研究奠定了基础。
