TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published...TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.展开更多
Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis soft...Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis softwares.The products of RT-PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC-S was verified and analyzed by corresponding restriction endonuclease(RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid,which was identified as S gene from Chinese isolate of TGEV.展开更多
A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length ...A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length was amplified by RT-PCR with special primers from the ZJ971 viral isolate of (IBV) and cloned into plasmid pBluescript SK( + ). The recombinants containing S gene of IBV-ZJ971 isolate were identified by digestion of restriction enzyme EcoRI, BamHI and PCR amplification. The cloned S gene from isolate IBV-7J971 was composed of 3492 bp in length encoding for a polypeptide of 1080 amino acids. Comparing the nucleotide of S gene of IBV isolate ZJ971 with that of reported IBV strains Beaudette, M41, Ark99 and CuT2, the homology was 97.3%, 97.5%, 88.6% and 85.6%, respectively; and the homology of the deduced amino acids of S protein of IBV isolate ZJ971 was 96%, 96.3%, 86.1% and 83.1% respectively; especially, the mutation of 3241st nucleotide of S gene of IBV isolate ZJ971 from G to T resulted in the translating termination of S protein at 3240th nucleotide site.展开更多
Objective: To study the relationship of the mutation of HBV preS/S gene in HBsAg carrying pregnant women and intrauterine transmission. Methods: Polymerase chain reaction (PCR) was used to amplify HBV preS/S gene from...Objective: To study the relationship of the mutation of HBV preS/S gene in HBsAg carrying pregnant women and intrauterine transmission. Methods: Polymerase chain reaction (PCR) was used to amplify HBV preS/S gene from sera of 8 HBsAg carrying pregnant women, 4 women's neonates infected with HBV, and the other's neonates non-infected with. The PCR products were cloned and 5 clones were chosen from every woman for DNA sequencing. Results: Heterogeneity of HBV preS/S gene in HBsAg carrying pregnant women having intrauterine transmission was much higher than that from having not intrauterine transmission, and the divergence rate of nucleotide sequences also higher strikingly. Conclusion: High heterogeneity of HBV preS/S gene of HBsAg positive pregnant women may be relative to high rate of intrauterine transmis-sion展开更多
INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-...INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.展开更多
为了研究仔猪腹泻的病原,试验采用了猪髋动脉内皮细胞（PIEC）对流行病学调查中检测到的3份猪传染性胃肠炎病毒（TGEV）阳性病料进行了培养、连续传代、PCR扩增、测序以及序列分析。结果表明：经过5代以后可以分离到1株出现典型细胞病...为了研究仔猪腹泻的病原,试验采用了猪髋动脉内皮细胞（PIEC）对流行病学调查中检测到的3份猪传染性胃肠炎病毒（TGEV）阳性病料进行了培养、连续传代、PCR扩增、测序以及序列分析。结果表明：经过5代以后可以分离到1株出现典型细胞病变的病毒,其TCID50可达1×10-5.33/0.1 m L,经过多种病原PCR检测证实该病毒为TGEV,并命名为T04株;对其S基因进行测序和分析,可知T04与TGEV其他毒株的核苷酸同源性为96.7%-99.8%,氨基酸的同源性为96.0%-99.5%。说明该分离毒株为典型的TGEV毒株,且从进化树可以看出T04与FJ株的同源性最高。展开更多
为建立猪传染性胃肠炎病毒(TGEV)SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据GenBank收录的TGEV-Miller毒株的S基因序列,设计合成1对特异性引物,用RT-PCR方法从疫苗株中扩增TGEV S基因的部分保守片段,并克隆到pGEM-T Easy载体上,得到重...为建立猪传染性胃肠炎病毒(TGEV)SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据GenBank收录的TGEV-Miller毒株的S基因序列,设计合成1对特异性引物,用RT-PCR方法从疫苗株中扩增TGEV S基因的部分保守片段,并克隆到pGEM-T Easy载体上,得到重组质粒作为荧光定量RT-PCR检测的标准模板,进行SYBR GreenⅠ荧光定量RT-PCR扩增,并制作标准曲线,建立TGEV的荧光定量RT-PCR检测方法。结果表明:该方法检测灵敏度可达30拷贝/μL,与猪圆环病毒、猪乙型脑炎病毒、猪伪狂犬病毒、猪瘟病毒和猪流感病毒和猪细小病毒不发生交叉反应,具有重复性好、特异性强、灵敏度高等优点,可用于临床TGEV感染的早期诊断以及分子流行病学调查。展开更多
文摘TH-98 isolate of transmissible gastroenteritis virus (TGEV) was propagated and harvested on swine testicle (ST) monolayer cell. Two pairs of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo version 4.1 and DNasis software. The products of PCR were named Sa and Sb, of 2.3 kb and 2.1 kb respectively. Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I multiple cloning sites of the same pUC18 plasmid. The recombinant pUC-S plasmid was identified and analyzed by corresponding restriction endonuclease and nested PCR on the basis of the genetic sites of S gene and pUC18 plasmid, which was identified as S gene of TGEV. Recombinant pUC-S was sequenced and analyzed in comparison with the other strains. Gene sequence comparison indicated that TH-98 shared 99, 97, 98, 97 and 94% identities with Purdue-115(US), Miller(US), TO14(Japan), FS772(British), 96-1933(British), respectively, their deduced amino acid homology was 99, 97, 97, 96 and 93% correspondingly. In addition, the analysis report verified that pUC-S owned a complete open reading frame (ORF) including initiation codon, signal sequences, remaining sequences and termination codon as well. Therefore, the results affirmed that S gene of TGEV TH-98 was extremely conservative.
文摘Chinese isolate of transmissible gastroenteritis virus(TGEV)was propagated and harvested in swine testicle(ST)cells.Two pairs of primers were designed according to the published sequence with Oligo 4.1 and DNasis softwares.The products of RT-PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoR I and Kpn I sites after Sb was cloned in Kpn I and Pst I sites of the same pUC18 plasmid.The recombinant designated pUC-S was verified and analyzed by corresponding restriction endonuclease(RE)and nested PCR on the basis of genetic sites of S gene and physical map of pUC18 plasmid,which was identified as S gene from Chinese isolate of TGEV.
基金the National NatureScience Foundation of China(30070570)the NatureScience Foundation of Zhejiang Province(399411) the Science and Technology Commission of Zhejiang Province(991102030).
文摘A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length was amplified by RT-PCR with special primers from the ZJ971 viral isolate of (IBV) and cloned into plasmid pBluescript SK( + ). The recombinants containing S gene of IBV-ZJ971 isolate were identified by digestion of restriction enzyme EcoRI, BamHI and PCR amplification. The cloned S gene from isolate IBV-7J971 was composed of 3492 bp in length encoding for a polypeptide of 1080 amino acids. Comparing the nucleotide of S gene of IBV isolate ZJ971 with that of reported IBV strains Beaudette, M41, Ark99 and CuT2, the homology was 97.3%, 97.5%, 88.6% and 85.6%, respectively; and the homology of the deduced amino acids of S protein of IBV isolate ZJ971 was 96%, 96.3%, 86.1% and 83.1% respectively; especially, the mutation of 3241st nucleotide of S gene of IBV isolate ZJ971 from G to T resulted in the translating termination of S protein at 3240th nucleotide site.
基金Supported by the National Natural Science Foundation of China (No. 39970652)
文摘Objective: To study the relationship of the mutation of HBV preS/S gene in HBsAg carrying pregnant women and intrauterine transmission. Methods: Polymerase chain reaction (PCR) was used to amplify HBV preS/S gene from sera of 8 HBsAg carrying pregnant women, 4 women's neonates infected with HBV, and the other's neonates non-infected with. The PCR products were cloned and 5 clones were chosen from every woman for DNA sequencing. Results: Heterogeneity of HBV preS/S gene in HBsAg carrying pregnant women having intrauterine transmission was much higher than that from having not intrauterine transmission, and the divergence rate of nucleotide sequences also higher strikingly. Conclusion: High heterogeneity of HBV preS/S gene of HBsAg positive pregnant women may be relative to high rate of intrauterine transmis-sion
文摘INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.
文摘为了研究仔猪腹泻的病原,试验采用了猪髋动脉内皮细胞（PIEC）对流行病学调查中检测到的3份猪传染性胃肠炎病毒（TGEV）阳性病料进行了培养、连续传代、PCR扩增、测序以及序列分析。结果表明：经过5代以后可以分离到1株出现典型细胞病变的病毒,其TCID50可达1×10-5.33/0.1 m L,经过多种病原PCR检测证实该病毒为TGEV,并命名为T04株;对其S基因进行测序和分析,可知T04与TGEV其他毒株的核苷酸同源性为96.7%-99.8%,氨基酸的同源性为96.0%-99.5%。说明该分离毒株为典型的TGEV毒株,且从进化树可以看出T04与FJ株的同源性最高。
