Full gene sequence of RNA-dependent RNA polymerase （RdRp） from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China （Zhejiang01/CHN/2002） was cloned. The sequence was 1 920 nucleotides in le...Full gene sequence of RNA-dependent RNA polymerase （RdRp） from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China （Zhejiang01/CHN/2002） was cloned. The sequence was 1 920 nucleotides in length coding 639 amino acid residues. Sequences comparison of RdRp showed Zhejiang01/CHN/2002 was 99.7% nucleotide sequence and 99.1% amino acids sequence homology with Japanese strain. The RdRp sequence was aligned with 8 representative picorna（-like） viruses and 8 highly conserved regions were detected. The result indicated their relevance function. Phylogenetic tree of 14 picorna（-like） viruses which RdRp presumed protein sequences revealed that the viruses from Iflavirus genus formed an independent clade. The RdRp was successfully expressed in BmN cells using Bac-to-Bac 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
The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SAR...The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SARS-CoV-2 is inevitable.The newly discovered coronavirus 2019(COVID-19)is a positive sense,single-stranded RNA,and enveloped virus,assigned to the beta CoV genus.The virus(SARS-CoV-2)is more infectious than the previously detected coronaviruses(MERS and SARS).Findings from many studies have revealed that S protein and RdRp are good targets for drug repositioning,novel therapeutic development(antibodies and small molecule drugs),and vaccine discovery.Therapeutics such as chloroquine,convalescent plasma,monoclonal antibodies,spike binding peptides,and small molecules could alter the ability of S protein to bind to the ACE-2 receptor,and drugs such as remdesivir(targeting SARS-CoV-2 RdRp),favipir,and emetine could prevent SASR-CoV-2 RNA synthesis.The novel vaccines such as mRNA1273(Moderna),3LNP-mRNAs(Pfizer/BioNTech),and ChAdOx1-S(University of Oxford/Astra Zeneca)targeting S protein have proven to be effective in combating the present pandemic.Further exploration of the potential of S protein and RdRp is crucial in fighting the present pandemic.展开更多
Dear Editor, Coxsackievirus A16 （CV A16） and enterovirus 71 （EV71） are currently the two primary causative agents of hand- foot-and-mouth disease （HFMD） （Solomon et al., 2010; Mao et al., 2014）, threatening he...Dear Editor, Coxsackievirus A16 （CV A16） and enterovirus 71 （EV71） are currently the two primary causative agents of hand- foot-and-mouth disease （HFMD） （Solomon et al., 2010; Mao et al., 2014）, threatening health of children world- wide. They both belong to the Enterovirus genus of the Picornaviridae family, and have single-stranded positive- sense RNA genomes of about 7.5 kilobases （kb） in length. As with other positive-strand RNA viruses, the genome rep- lication process ofCV A16 is carried out by a membrane- associated replication complex with the virally encoded RNA-dependent RNA polymerase （RdRP） as the essential catalytic enzyme.展开更多
基金supported by the National 863 Program of China (2006AA10A119)the Key Project of Science and Technology Commission of Zhejiang Province,China (2003C22013)
文摘Full gene sequence of RNA-dependent RNA polymerase （RdRp） from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China （Zhejiang01/CHN/2002） was cloned. The sequence was 1 920 nucleotides in length coding 639 amino acid residues. Sequences comparison of RdRp showed Zhejiang01/CHN/2002 was 99.7% nucleotide sequence and 99.1% amino acids sequence homology with Japanese strain. The RdRp sequence was aligned with 8 representative picorna（-like） viruses and 8 highly conserved regions were detected. The result indicated their relevance function. Phylogenetic tree of 14 picorna（-like） viruses which RdRp presumed protein sequences revealed that the viruses from Iflavirus genus formed an independent clade. The RdRp was successfully expressed in BmN cells using Bac-to-Bac 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.
文摘The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SARS-CoV-2 is inevitable.The newly discovered coronavirus 2019(COVID-19)is a positive sense,single-stranded RNA,and enveloped virus,assigned to the beta CoV genus.The virus(SARS-CoV-2)is more infectious than the previously detected coronaviruses(MERS and SARS).Findings from many studies have revealed that S protein and RdRp are good targets for drug repositioning,novel therapeutic development(antibodies and small molecule drugs),and vaccine discovery.Therapeutics such as chloroquine,convalescent plasma,monoclonal antibodies,spike binding peptides,and small molecules could alter the ability of S protein to bind to the ACE-2 receptor,and drugs such as remdesivir(targeting SARS-CoV-2 RdRp),favipir,and emetine could prevent SASR-CoV-2 RNA synthesis.The novel vaccines such as mRNA1273(Moderna),3LNP-mRNAs(Pfizer/BioNTech),and ChAdOx1-S(University of Oxford/Astra Zeneca)targeting S protein have proven to be effective in combating the present pandemic.Further exploration of the potential of S protein and RdRp is crucial in fighting the present pandemic.
文摘Dear Editor, Coxsackievirus A16 （CV A16） and enterovirus 71 （EV71） are currently the two primary causative agents of hand- foot-and-mouth disease （HFMD） （Solomon et al., 2010; Mao et al., 2014）, threatening health of children world- wide. They both belong to the Enterovirus genus of the Picornaviridae family, and have single-stranded positive- sense RNA genomes of about 7.5 kilobases （kb） in length. As with other positive-strand RNA viruses, the genome rep- lication process ofCV A16 is carried out by a membrane- associated replication complex with the virally encoded RNA-dependent RNA polymerase （RdRP） as the essential catalytic enzyme.
