目的 克隆人腺病毒 3型 (Ad3)六邻体 - L 1(Hexon- L 1)、六邻体 - L 2 (Hexon- L 2 )区基因 ,构建含有该基因的重组质粒 ,并进行测序鉴定 ,为进一步构建表达载体 ,制备基因工程疫苗打下基础。方法 从 Ad3感染的 He L a细胞中提取 Ad3...目的 克隆人腺病毒 3型 (Ad3)六邻体 - L 1(Hexon- L 1)、六邻体 - L 2 (Hexon- L 2 )区基因 ,构建含有该基因的重组质粒 ,并进行测序鉴定 ,为进一步构建表达载体 ,制备基因工程疫苗打下基础。方法 从 Ad3感染的 He L a细胞中提取 Ad3DNA,用 PCR方法扩增 Hexon- L 1、Hexon- L 2基因 ,将得到的片段定向克隆到克隆载体 p U C19质粒中 ,对克隆片段进行 DNA测序鉴定。结果 构建了重组质粒 p UC19Hexon,测序结果与 Genebank中该序列进行同源性比较证明克隆的片段为 Ad3六邻体序列。结论 成功克隆了 Ad3的 Hexon- L 1、Hexon- L 2区基因。展开更多
The plasmid PE which harbored the whole hexon-encoding gene of egg drop syndrome virus(EDSV) was identified and the hexon protein gene was obtained from it by restriction endonucleases.The gene was then directionally ...The plasmid PE which harbored the whole hexon-encoding gene of egg drop syndrome virus(EDSV) was identified and the hexon protein gene was obtained from it by restriction endonucleases.The gene was then directionally inserted into the PphI/KpnI sites of pQE32 and fused with the 6×His gene.This recombinant plasmid was transformed into E.coli M15 for expression and induced with IPTG.It was demonstrated by SDS-PAGE and Western blot that one expressed protein,110kD in size,could be specifically recognized by polyclonal anti-EDSV serum.The objective product was principally soluble and accounted for 21% of the total cellular proteins.The protein was used to detect the existence of antiEDSV IgG in 41 clinical chicken sera by agar diffusion test,and the result was in accordance with that when EDSV was used as antigen.These results showed that the expressed hexon recombinant protein can be used as diagnostic antigen of EDSV.展开更多
According to the nucleotide sequence of egg drop syndrome virus(EDSV)AV-127 strain (a foreign standard isolate)from the GenBank,a pair of oligonucleotide primers was designed and synthesizedWith use of polymerase ch...According to the nucleotide sequence of egg drop syndrome virus(EDSV)AV-127 strain (a foreign standard isolate)from the GenBank,a pair of oligonucleotide primers was designed and synthesizedWith use of polymerase chain reaction(PCR),the hexon-encoding gene fragment was amplified from the genome of EDSV SG9301 strain that was isolated from Sichuan Province of China and the amplified fragment was directly inserted into pMD-T vectorThe recombinant plasmid harboring the hexon-encoding gene was identified by digestion of restriction endonucleases and PCRThen,a positive clone was sequencedThe result showed that the recombinant plasmid contained the complete sequence of the hexon-encoding gene and the hexon-encoding gene was 2733 base pair long which encoded 910 amino acids, identical with AV-127 strain and AAV-2 strain (an attenuated strain)Comparison with AV-127 strain indicated that there was 9985% homology at the nucleotide levelThe homology of the deduced amino acid sequence with AV-127 strain and AAv-2 strain was over 98%,which indicated that the hexon protein was conservativeBut comparatively,the homology of the hexon protein of SG9301 strain with AV-127 strain was higher(9978%),the homology of the hexon protein of AAV-2 strain with AV-127 strain and SG9301 strain were 9868% and 9846% respectivelyComparison of nucleotides and amino acids suggested that the biological character is consistent with the homologies of nucleotide and amino acid of the hexon(Namely,the homology of the hexon of virulence strain with virulence strain was higher than that of virulence strain with attenuated strain),the hexon was one of the most conserved viral proteins among EDSV展开更多
[目的]分析2004年夏广州市某幼儿园流行的3型腺病毒(adenovirus type 3,Ad3)的六邻体基因序列,为 Ad3的基础及临床研究提供依据。[方法]采集急性咽结膜炎患儿急性期的眼、咽拭子,提取腺病毒DNA,分两段进行 PCR,扩增腺病毒六邻体基因,将...[目的]分析2004年夏广州市某幼儿园流行的3型腺病毒(adenovirus type 3,Ad3)的六邻体基因序列,为 Ad3的基础及临床研究提供依据。[方法]采集急性咽结膜炎患儿急性期的眼、咽拭子,提取腺病毒DNA,分两段进行 PCR,扩增腺病毒六邻体基因,将扩增的片断进行T-载体克隆及序列分析。[结果]成功地测出了Ad3六邻体基因序列,并在Genebank登记注册,注册号AY878716,与Genebank中Ad3六邻体基因序列进行同源性比较,同源性为99％。 [结论]腺病毒(Ad3)六邻体基因序列,并与(Genebank中Ad3六邻体基因序列变异是否与其毒力的改变和致病性、传染性相关,尚需作进一步的研究探索。展开更多
文摘目的 克隆人腺病毒 3型 (Ad3)六邻体 - L 1(Hexon- L 1)、六邻体 - L 2 (Hexon- L 2 )区基因 ,构建含有该基因的重组质粒 ,并进行测序鉴定 ,为进一步构建表达载体 ,制备基因工程疫苗打下基础。方法 从 Ad3感染的 He L a细胞中提取 Ad3DNA,用 PCR方法扩增 Hexon- L 1、Hexon- L 2基因 ,将得到的片段定向克隆到克隆载体 p U C19质粒中 ,对克隆片段进行 DNA测序鉴定。结果 构建了重组质粒 p UC19Hexon,测序结果与 Genebank中该序列进行同源性比较证明克隆的片段为 Ad3六邻体序列。结论 成功克隆了 Ad3的 Hexon- L 1、Hexon- L 2区基因。
文摘The plasmid PE which harbored the whole hexon-encoding gene of egg drop syndrome virus(EDSV) was identified and the hexon protein gene was obtained from it by restriction endonucleases.The gene was then directionally inserted into the PphI/KpnI sites of pQE32 and fused with the 6×His gene.This recombinant plasmid was transformed into E.coli M15 for expression and induced with IPTG.It was demonstrated by SDS-PAGE and Western blot that one expressed protein,110kD in size,could be specifically recognized by polyclonal anti-EDSV serum.The objective product was principally soluble and accounted for 21% of the total cellular proteins.The protein was used to detect the existence of antiEDSV IgG in 41 clinical chicken sera by agar diffusion test,and the result was in accordance with that when EDSV was used as antigen.These results showed that the expressed hexon recombinant protein can be used as diagnostic antigen of EDSV.
文摘According to the nucleotide sequence of egg drop syndrome virus(EDSV)AV-127 strain (a foreign standard isolate)from the GenBank,a pair of oligonucleotide primers was designed and synthesizedWith use of polymerase chain reaction(PCR),the hexon-encoding gene fragment was amplified from the genome of EDSV SG9301 strain that was isolated from Sichuan Province of China and the amplified fragment was directly inserted into pMD-T vectorThe recombinant plasmid harboring the hexon-encoding gene was identified by digestion of restriction endonucleases and PCRThen,a positive clone was sequencedThe result showed that the recombinant plasmid contained the complete sequence of the hexon-encoding gene and the hexon-encoding gene was 2733 base pair long which encoded 910 amino acids, identical with AV-127 strain and AAV-2 strain (an attenuated strain)Comparison with AV-127 strain indicated that there was 9985% homology at the nucleotide levelThe homology of the deduced amino acid sequence with AV-127 strain and AAv-2 strain was over 98%,which indicated that the hexon protein was conservativeBut comparatively,the homology of the hexon protein of SG9301 strain with AV-127 strain was higher(9978%),the homology of the hexon protein of AAV-2 strain with AV-127 strain and SG9301 strain were 9868% and 9846% respectivelyComparison of nucleotides and amino acids suggested that the biological character is consistent with the homologies of nucleotide and amino acid of the hexon(Namely,the homology of the hexon of virulence strain with virulence strain was higher than that of virulence strain with attenuated strain),the hexon was one of the most conserved viral proteins among EDSV
