Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isola...Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isolates [65 con-genitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. Results Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 se-quences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.展开更多
目的探讨长散在核元件1读码框2基因(L1-ORF2)对GES-1细胞衰老的影响及分子调控机制。方法采用高糖诱导法构建GES-1细胞衰老模型,构建L1-ORF2 si RNA载体,脂质体法将其瞬时转染正常及衰老的GES-1细胞,转染48h后用细胞计数法绘制细胞生长...目的探讨长散在核元件1读码框2基因(L1-ORF2)对GES-1细胞衰老的影响及分子调控机制。方法采用高糖诱导法构建GES-1细胞衰老模型,构建L1-ORF2 si RNA载体,脂质体法将其瞬时转染正常及衰老的GES-1细胞,转染48h后用细胞计数法绘制细胞生长曲线,流式细胞仪分析细胞周期,β-半乳糖苷酶染色检测细胞衰老情况,Western blotting检测转染细胞中L1-ORF2、P53、P21蛋白表达水平。结果成功构建了稳定的GES-1细胞衰老模型及L1-ORF2si RNA载体。与转染了阴性对照载体的细胞相比,转染L1-ORF2 si RNA载体的正常及衰老GES-1细胞的L1-ORF2表达均下降(P<0.05)。与转染阴性对照载体的衰老GES-1细胞相比,转染L1-ORF2 si RNA载体的衰老GES-1细胞增殖速度变快(P<0.05),G0/G1期比例明显减少(34.2%vs 39.3%,P<0.05),β-半乳糖苷酶染色比例下降(56%vs 69%,P<0.05),而转染阴性对照载体和L1-ORF2 si RNA载体的正常GES-1细胞相比则无明显差异(P>0.05)。P53蛋白仅在衰老GES-1细胞中有表达,在正常GES-1细胞中无表达,而P21蛋白在正常和衰老的GES-1细胞中均有表达,且后者表达更高(P<0.05)。与转染阴性对照载体的细胞相比,转染L1-ORF2 si RNA载体的GES-1细胞P53、P21蛋白表达量明显下降(P<0.05)。结论L1-ORF2 si RNA载体可使正常及衰老的GES-1细胞中L1-ORF2表达下调,促进衰老GES-1细胞的生长和增殖,而对正常GES-1细胞无明显影响。P53、P21蛋白参与了L1-ORF2调控细胞衰老的过程。展开更多
目的探讨戊型肝炎病毒(hepatitis E virus,HEV)ORF2基因在人胚肾293细胞的表达。方法利用聚合酶链反应(PCR)技术,以HEV ORF2为模板扩增其部分基因片段,克隆入真核表达载体pcDNA 3．1中,构建真核表达载体质粒,转染人胚肾293细胞,用G418(G...目的探讨戊型肝炎病毒(hepatitis E virus,HEV)ORF2基因在人胚肾293细胞的表达。方法利用聚合酶链反应(PCR)技术,以HEV ORF2为模板扩增其部分基因片段,克隆入真核表达载体pcDNA 3．1中,构建真核表达载体质粒,转染人胚肾293细胞,用G418(Geneticin,遗传霉素)加压筛选得到稳定表达株。用western-blot方法对表达产物进行免疫学特性初步研究。结果含ORF2结构基因部分片段的重组质粒在人胚肾293细胞中得到表达。结论表达的ORF2重组蛋白具有抗HEV抗体识别的抗原表位和一定的抗原性,为进一步研制HEV的诊断试剂奠定了基础。展开更多
基金Supported by the National Natural Science Foundation of China (30170986).
文摘Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isolates [65 con-genitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. Results Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 se-quences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.
文摘目的探讨长散在核元件1读码框2基因(L1-ORF2)对GES-1细胞衰老的影响及分子调控机制。方法采用高糖诱导法构建GES-1细胞衰老模型,构建L1-ORF2 si RNA载体,脂质体法将其瞬时转染正常及衰老的GES-1细胞,转染48h后用细胞计数法绘制细胞生长曲线,流式细胞仪分析细胞周期,β-半乳糖苷酶染色检测细胞衰老情况,Western blotting检测转染细胞中L1-ORF2、P53、P21蛋白表达水平。结果成功构建了稳定的GES-1细胞衰老模型及L1-ORF2si RNA载体。与转染了阴性对照载体的细胞相比,转染L1-ORF2 si RNA载体的正常及衰老GES-1细胞的L1-ORF2表达均下降(P<0.05)。与转染阴性对照载体的衰老GES-1细胞相比,转染L1-ORF2 si RNA载体的衰老GES-1细胞增殖速度变快(P<0.05),G0/G1期比例明显减少(34.2%vs 39.3%,P<0.05),β-半乳糖苷酶染色比例下降(56%vs 69%,P<0.05),而转染阴性对照载体和L1-ORF2 si RNA载体的正常GES-1细胞相比则无明显差异(P>0.05)。P53蛋白仅在衰老GES-1细胞中有表达,在正常GES-1细胞中无表达,而P21蛋白在正常和衰老的GES-1细胞中均有表达,且后者表达更高(P<0.05)。与转染阴性对照载体的细胞相比,转染L1-ORF2 si RNA载体的GES-1细胞P53、P21蛋白表达量明显下降(P<0.05)。结论L1-ORF2 si RNA载体可使正常及衰老的GES-1细胞中L1-ORF2表达下调,促进衰老GES-1细胞的生长和增殖,而对正常GES-1细胞无明显影响。P53、P21蛋白参与了L1-ORF2调控细胞衰老的过程。
文摘目的探讨戊型肝炎病毒(hepatitis E virus,HEV)ORF2基因在人胚肾293细胞的表达。方法利用聚合酶链反应(PCR)技术,以HEV ORF2为模板扩增其部分基因片段,克隆入真核表达载体pcDNA 3．1中,构建真核表达载体质粒,转染人胚肾293细胞,用G418(Geneticin,遗传霉素)加压筛选得到稳定表达株。用western-blot方法对表达产物进行免疫学特性初步研究。结果含ORF2结构基因部分片段的重组质粒在人胚肾293细胞中得到表达。结论表达的ORF2重组蛋白具有抗HEV抗体识别的抗原表位和一定的抗原性,为进一步研制HEV的诊断试剂奠定了基础。
