戊型肝炎病毒(Hepatitis E virus,HEV)是造成急性肝炎最常见的原因之一。HEV基因组由5′非编码区、3个开放阅读框(ORF1、ORF2、ORF3)和3′非编码区组成,仅在HEV-1中发现了ORF4,并与ORF1重叠。各种编码蛋白在HEV的复制和感染中发挥着不...戊型肝炎病毒(Hepatitis E virus,HEV)是造成急性肝炎最常见的原因之一。HEV基因组由5′非编码区、3个开放阅读框(ORF1、ORF2、ORF3)和3′非编码区组成,仅在HEV-1中发现了ORF4,并与ORF1重叠。各种编码蛋白在HEV的复制和感染中发挥着不同的作用,而HEV的复制是由ORF1编码的RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase,RdRp)所介导的。HEV RdRp是由多个蛋白亚基组成的复合酶,具有7个保守基序,这些保守基序在RNA合成过程中发挥核苷酸识别、合成、延伸、修饰和稳定等作用,保证了RdRp的功能,在HEV的复制和转录中起到关键作用。因此,以RdRp作为抗HEV药物作用靶点的治疗方案具有很好的应用前景,是目前药物开发的一种主流思路。目前,已发现利巴韦林、索非布韦、2′-C-甲基胞苷(2CMC)等核苷类RdRp抑制剂和锌、GPC-N114等非核苷类RdRp抑制剂对HEV有较强的抑制作用,可作为潜在的抗HEV药物进行深入研究。笔者对HEV编码蛋白和HEV RdRp的结构与功能进行阐述,总结目前发现的对HEV有抑制作用的RdRp抑制剂,以期为HEV的药物开发提供一种新的思路。展开更多
A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restr...A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.展开更多
Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA...Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA replication. It is composed of four subunits, making it the simplest multisubunit RNAP known. Two subunits contain motifs found at the catalytic center of other RNAPs and a third has capping enzyme functions. The function of the fourth subunit is not known. Structural studies on this unique RNAP will provide new insights into the functions of this enzyme and the regulation of viral genes and may be instrumental to optimize the baculovirus gene expression system.展开更多
目的探讨黄曲霉(A.flavus)基因敲除体系的构建和RNA依赖的RNA聚合酶1(RDRP1)基因在A.flavus生长发育中的作用。方法通过National Center for Biotechnology Information网址查找RDRP1基因,设计上下游序列引物,引入融合片段20 bp,采用重...目的探讨黄曲霉(A.flavus)基因敲除体系的构建和RNA依赖的RNA聚合酶1(RDRP1)基因在A.flavus生长发育中的作用。方法通过National Center for Biotechnology Information网址查找RDRP1基因,设计上下游序列引物,引入融合片段20 bp,采用重叠PCR(overlap PCR)法融合RDRP1基因上下游片段和嘧啶磺胺抗性基因(ptrA);采用聚乙二醇(PEG)介导方法将该融合片段导入A.flavus的原生质体中获得RDRP1阳性转化子(ptrA抗性),采用Sourthern blot鉴定筛选RDRP1基因突变菌株;对RDRP1基因突变菌株,采用十字交叉法测定生长速率、血细胞计数板统计产孢量,手动计数Wickerham Medium(WKM)+尿嘧啶尿苷(UU)培养基上产生的菌核数量。结果获得ptrA抗性转化子13个;Sourthern blot鉴定4个为RDRP1基因缺失菌株,效率30.8%;与CA14相比,RDRP1基因突变菌株在表型、生长率、产孢量及菌核发育上差异无统计学意义(P>0.05)。结论overlap PCR结合PEG介导转化的方式可短时间内获得A.flavus基因敲除突变菌株,RDRP1基因不参与A.flavus表型、生长率、产孢量及菌核发育的调控作用。展开更多
An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was trans...An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.展开更多
目的构建含T7RNA聚合酶(T7RNA Polymerase,T7RNP)的真核表达载体,同时构建含T7启动子、增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和T7RNP转录终止信号的载体,并通过观察EGFP的表达推测T7RNP表达和T7表达系统介导基...目的构建含T7RNA聚合酶(T7RNA Polymerase,T7RNP)的真核表达载体,同时构建含T7启动子、增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)和T7RNP转录终止信号的载体,并通过观察EGFP的表达推测T7RNP表达和T7表达系统介导基因转录的功能。方法根据编码T7RNP的基因序列,应用PCR技术从含有T7RNP基因的E.coli BL21(DE3)基因组中扩增编码T7RNP的基因片段,克隆至载体pcDNA3·1(+)。同时从px8δt载体和pGEM-Teasy/EGFP上切下T7RNP识别的终止信号(terminator,TER)和编码EGFP的基因,克隆至载体pcD-NAII。将获得的两个重组质粒共转染BHK细胞。48h后,通过观察EGFP基因在真核细胞内的表达情况,推测T7RNP基因的表达及表达后识别T7启动子介导基因转录功能。结果成功构建了含T7RNP编码基因的真核表达载体pcDNA3·1(+)/T7RNP和原核表达载体pcDNAII/EG-FP/TER,共转染BHK细胞后,可观察到EGFP表达的绿色荧光。结论T7RNP能顺利在真核细胞内表达,并通过与T7启动子和TER的相互作用,成功实现了EGFP基因的转录和表达。展开更多
文摘A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.
文摘Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA replication. It is composed of four subunits, making it the simplest multisubunit RNAP known. Two subunits contain motifs found at the catalytic center of other RNAPs and a third has capping enzyme functions. The function of the fourth subunit is not known. Structural studies on this unique RNAP will provide new insights into the functions of this enzyme and the regulation of viral genes and may be instrumental to optimize the baculovirus gene expression system.
文摘An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.
