采用传统纯培养方法对内蒙古不同地区的12份绵羊奶油样品中的乳酸菌进行分离纯化,运用16S r DNA序列分析方法进行属种鉴定,同时采用实时荧光定量聚合酶链反应(q-PCR)技术对包头地区样品中优势菌群数量进行了定量研究。分离鉴定结果表明...采用传统纯培养方法对内蒙古不同地区的12份绵羊奶油样品中的乳酸菌进行分离纯化,运用16S r DNA序列分析方法进行属种鉴定,同时采用实时荧光定量聚合酶链反应(q-PCR)技术对包头地区样品中优势菌群数量进行了定量研究。分离鉴定结果表明三个地区绵羊发酵奶油中分离鉴定的44株乳酸菌,其中Lactococcus lactis subsp.Lactis为优势菌群。q-PCR定量结果表明3种优势菌属的数量关系为Lac.Lactis.subsp.lactis>L.plantarum>Leu.Mesenteroides。展开更多
AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting ...AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR)method and the restriction enzyme reaction.In vitro RNA transcripts of chimera,prototype J6JFH and negative control J6JFH1(GND)were prepared and transfected into Huh-7.5 cells with liposomes.Immunofluorescence assay(IFA),fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS:The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point(2.58 ±5.97×106 vs 4.27±1.72×104,P=0.032).The maximal level of HCV RNA in chimera was 5.6±1.8× 104 GE/μg RNA at day 34 after transfection,while the wild type reached a peak level at day 13 which was 126 folds higher(70.65±14.11×105 vs 0.56±0.90 ×105,P=0.028).HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level.Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles,ranging from 10±5 ffu/mL to 78.3±23.6 ffu/mL,while that of FL-J6JFH1 ranged from 5.8±1.5×102 ffu/mL to 2.5±1.4×104 ffu/mL.CONCLUSION:JFH1 NS5A might play an important role in the robust replication of J6JFH1.The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.展开更多
DNA甲基化主要是通过在甲基转移酶的催化下修饰基因组DNA来响应外界环境的胁迫,是表观遗传学的重要手段之一,参与植物中DNA甲基化起始的主要DNA甲基转移酶是DRM2。文章通过美国国家生物技术信息中心(National Center for Biotechnology ...DNA甲基化主要是通过在甲基转移酶的催化下修饰基因组DNA来响应外界环境的胁迫,是表观遗传学的重要手段之一,参与植物中DNA甲基化起始的主要DNA甲基转移酶是DRM2。文章通过美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库,在番茄中发现了一个与拟南芥胞嘧啶甲基转移酶DRM2同源性较高的序列,即SlDRM2L。通过GenBank(登录号NM-001246974)获得番茄DNA甲基转移酶SlDRM2L基因的cDNA全长序列。实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)结果表明SlDRM2L在所有组织中均有表达,叶和花的表达量最高。同时利用烟草瞬时表达系统将构建的绿色荧光瞬时表达载体35S::SlDRM2L-GFP用于亚细胞定位实验,通过激光共聚焦显微镜观察可知SlDRM2L蛋白定位于细胞核中;此外,在高温条件下(39℃,4 h),番茄SlDRM2L的基因转录水平明显提高,表明SlDRM2L介导的DNA甲基化可能受高温诱导。该文为发现SlDRM2L在植物生长发育中的作用提供了相关的科学依据,为深入研究SlDRM2L的功能奠定了理论基础。展开更多
文摘采用传统纯培养方法对内蒙古不同地区的12份绵羊奶油样品中的乳酸菌进行分离纯化,运用16S r DNA序列分析方法进行属种鉴定,同时采用实时荧光定量聚合酶链反应(q-PCR)技术对包头地区样品中优势菌群数量进行了定量研究。分离鉴定结果表明三个地区绵羊发酵奶油中分离鉴定的44株乳酸菌,其中Lactococcus lactis subsp.Lactis为优势菌群。q-PCR定量结果表明3种优势菌属的数量关系为Lac.Lactis.subsp.lactis>L.plantarum>Leu.Mesenteroides。
基金Supported by The Natural Science Foundation of China,No. 30872247 and 30600529the PLA medical research funds of China,No. 06H021 and 06Z027 and Shanghai LAD Project (B901)
文摘AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR)method and the restriction enzyme reaction.In vitro RNA transcripts of chimera,prototype J6JFH and negative control J6JFH1(GND)were prepared and transfected into Huh-7.5 cells with liposomes.Immunofluorescence assay(IFA),fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS:The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point(2.58 ±5.97×106 vs 4.27±1.72×104,P=0.032).The maximal level of HCV RNA in chimera was 5.6±1.8× 104 GE/μg RNA at day 34 after transfection,while the wild type reached a peak level at day 13 which was 126 folds higher(70.65±14.11×105 vs 0.56±0.90 ×105,P=0.028).HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level.Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles,ranging from 10±5 ffu/mL to 78.3±23.6 ffu/mL,while that of FL-J6JFH1 ranged from 5.8±1.5×102 ffu/mL to 2.5±1.4×104 ffu/mL.CONCLUSION:JFH1 NS5A might play an important role in the robust replication of J6JFH1.The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.
文摘DNA甲基化主要是通过在甲基转移酶的催化下修饰基因组DNA来响应外界环境的胁迫,是表观遗传学的重要手段之一,参与植物中DNA甲基化起始的主要DNA甲基转移酶是DRM2。文章通过美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库,在番茄中发现了一个与拟南芥胞嘧啶甲基转移酶DRM2同源性较高的序列,即SlDRM2L。通过GenBank(登录号NM-001246974)获得番茄DNA甲基转移酶SlDRM2L基因的cDNA全长序列。实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)结果表明SlDRM2L在所有组织中均有表达,叶和花的表达量最高。同时利用烟草瞬时表达系统将构建的绿色荧光瞬时表达载体35S::SlDRM2L-GFP用于亚细胞定位实验,通过激光共聚焦显微镜观察可知SlDRM2L蛋白定位于细胞核中;此外,在高温条件下(39℃,4 h),番茄SlDRM2L的基因转录水平明显提高,表明SlDRM2L介导的DNA甲基化可能受高温诱导。该文为发现SlDRM2L在植物生长发育中的作用提供了相关的科学依据,为深入研究SlDRM2L的功能奠定了理论基础。
