[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector a...[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30.展开更多
In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and r...In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains.A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene.The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels.Phylogenetic analysis showed three distinct clusters of SPPV,GTPV and Lumpy skin disease virus (LSDV) isolates.Further,multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I,which is present in SPPV isolates studied.This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains.The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV.The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.展开更多
【目的】对绵羊痘病毒甘肃古浪株RING finger蛋白的基因进行克隆,表达以及序列分析,初步探究绵羊痘病毒RING finger蛋白是否具有E3泛素连接酶活性,为阐明其在绵羊痘病毒感染过程中对泛素蛋白酶体系统的调控作用奠定基础.【方法】以绵羊...【目的】对绵羊痘病毒甘肃古浪株RING finger蛋白的基因进行克隆,表达以及序列分析,初步探究绵羊痘病毒RING finger蛋白是否具有E3泛素连接酶活性,为阐明其在绵羊痘病毒感染过程中对泛素蛋白酶体系统的调控作用奠定基础.【方法】以绵羊痘病毒甘肃古浪株DNA为模板,通过PCR扩增 RING finger 基因.利用Pfam数据库、DNAstar等软件进行序列及遗传进化分析;将 RING finger 基因片段插入原核表达载体pGEX-4T-1中,构建pGEX-SPPVRFP重组表达载体,在大肠杆菌BL21(DE3)中诱导表达,并进行SDS-PAGA和Western-blot分析.【结果】绵羊痘病毒 RING finger 基因由723个核苷酸组成,编码的240个氨基酸的分子量约为28.5 ku,具有RING finger结构域.不同羊痘病毒株间 RING finger 基因核苷酸序列同源性高达99.2%,氨基酸序列同源性高达98.3%.不同的RING finger蛋白都含有8个保守的半胱氨酸和组氨酸.SDS-PAGA分析显示,重组SPPVRFP大小约为55 ku,Western-blot分析显示,重组SPPVRFP不能与绵羊痘病毒阳性血清反应.【结论】成功克隆、表达并纯化了绵羊痘病毒 RING finger 基因,对SPPV GS-GL株RING-finger蛋白进行序列分析,推测其可能具有E3泛素连接酶活性.展开更多
基金Supported by the National Natural Science Foundation of China(31001056)the National Natural Science Foundation of China(31101802)+1 种基金Major Program for New Transgenic Organism Verities Breeding of Ministry of Agriculture of China(2009ZX08008-010B)Key Science and Technology Foundation of Gansu Province(092NKDA032)~~
文摘[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30.
文摘In the present study,the partial gene sequences of P32 protein,an immunogenic envelope protein of Capripoxviruses (CaPV),were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates,and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains.A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene.The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels.Phylogenetic analysis showed three distinct clusters of SPPV,GTPV and Lumpy skin disease virus (LSDV) isolates.Further,multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I,which is present in SPPV isolates studied.This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains.The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV.The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.
文摘【目的】对绵羊痘病毒甘肃古浪株RING finger蛋白的基因进行克隆,表达以及序列分析,初步探究绵羊痘病毒RING finger蛋白是否具有E3泛素连接酶活性,为阐明其在绵羊痘病毒感染过程中对泛素蛋白酶体系统的调控作用奠定基础.【方法】以绵羊痘病毒甘肃古浪株DNA为模板,通过PCR扩增 RING finger 基因.利用Pfam数据库、DNAstar等软件进行序列及遗传进化分析;将 RING finger 基因片段插入原核表达载体pGEX-4T-1中,构建pGEX-SPPVRFP重组表达载体,在大肠杆菌BL21(DE3)中诱导表达,并进行SDS-PAGA和Western-blot分析.【结果】绵羊痘病毒 RING finger 基因由723个核苷酸组成,编码的240个氨基酸的分子量约为28.5 ku,具有RING finger结构域.不同羊痘病毒株间 RING finger 基因核苷酸序列同源性高达99.2%,氨基酸序列同源性高达98.3%.不同的RING finger蛋白都含有8个保守的半胱氨酸和组氨酸.SDS-PAGA分析显示,重组SPPVRFP大小约为55 ku,Western-blot分析显示,重组SPPVRFP不能与绵羊痘病毒阳性血清反应.【结论】成功克隆、表达并纯化了绵羊痘病毒 RING finger 基因,对SPPV GS-GL株RING-finger蛋白进行序列分析,推测其可能具有E3泛素连接酶活性.
