旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blo...旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blot验证该重组蛋白能够与小鼠抗轮状病毒血清反应。将该重组蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性杂交瘤细胞,Western blot和间接免疫荧光验证阳性杂交瘤细胞分泌的单克隆抗体与RVA的反应性。经3轮亚克隆最终获得一株能稳定分泌针对VP6蛋白单克隆抗体的阳性杂交瘤细胞株5G9,其分泌的单抗亚类为IgG1,轻链类型为Kappa,制备的小鼠腹水效价可达1∶4096000,能够与不同基因型的RVA反应,同时不与常见的猪腹泻相关病毒反应,为未来猪RVA相关基础研究、临床血清学检测方法的开发奠定了基础。展开更多
为探究FILIP1L(filamin A-interacting protein 1-like)蛋白对猪流行性腹泻病毒(PEDV)的影响,以Vero细胞中提取的总RNA反转录的cDNA为模板,PCR扩增出FILIP1L基因,克隆到p3×FLAG-CMV-10真核表达载体上。过表达或沉默FILIP1L基因,通...为探究FILIP1L(filamin A-interacting protein 1-like)蛋白对猪流行性腹泻病毒(PEDV)的影响,以Vero细胞中提取的总RNA反转录的cDNA为模板,PCR扩增出FILIP1L基因,克隆到p3×FLAG-CMV-10真核表达载体上。过表达或沉默FILIP1L基因,通过免疫印迹和定量PCR检测FILIP1L蛋白的表达情况,并研究其抗病毒功能。结果:成功构建3×FLAG-CMV-10-FILIP1L重组质粒,转染后能够正常表达。PEDV感染Vero细胞后,FILIP1L基因的mRNA表达显著上调;过表达FILIP1L基因显著抑制PEDV的复制;下调FILIP1L的表达可显著促进PEDV的复制。综上,FILIP1L蛋白能够抑制PEDV在Vero细胞的复制,研究结果为寻找新的抗PEDV药物靶点和研发新型疫苗提供理论依据。展开更多
[Objective] This study aimed to investigate the genetic variation of g E gene of an epidemic pseudorabies virus（PRV） strain and its pathogenicity to piglets. [Method] By serial passage in Vero cells, a PRV strain wa...[Objective] This study aimed to investigate the genetic variation of g E gene of an epidemic pseudorabies virus（PRV） strain and its pathogenicity to piglets. [Method] By serial passage in Vero cells, a PRV strain was isolated from the brain tissues of stillborn fetuses delivered by sows with suspected PRV infection and preliminarily identified by PCR. g E gene of the isolated PRV strain was amplified and sequenced for phylogenetic analysis. In addition, the pathogenicity of the isolated PRV strain to 6-week-old piglets was evaluated. [Result] A PRV strain was successfully isolated and named PRV N5 B strain, which could proliferate in Vero cells and TCID50 of the 15 thgeneration virus liquid reached 10^7.125/0.1 ml. Specific bands could be amplified by PCR. g E gene in the isolated PRV strain was 1 740 bp in length. A phylogenetic tree was constructed based on full-length g E sequences, which showed that PRV N5 B strain and PRV strains isolated since 2012 were clustered into the same independent category and shared 99.7%-100% homology of nucleotide sequences. Compared with related sequences published previously, there were insertions of three consecutive bases at two loci. Animal experiments showed that intranasal inoculation of 6-week-old piglets with 2 ml of PRV N5 B strain（10^6/0.1 ml） led to a mortality rate of 100%. [Conclusion] In this study,genetic variability of g E gene in PRV N5 B isolate and its pathogenicity to piglets were analyzed, which provided a theoretical basis for the development of new vaccines to prevent and control porcine pseudorabies.展开更多
该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;...该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;以阳性质粒为模板,建立SYBR Green Ⅰ荧光定量PCR检测方法,并进行敏感性和特异性检测。经测序证实扩增片段属于P1,所建立的SYBR Green Ⅰ荧光定量PCR检测P1的反应在101-108拷贝/μL之间具有良好的线性关系,反应的检出下限为10拷贝/μL,而对猪伪狂犬病病毒、猪细小病毒、猪繁殖与呼吸综合征病毒等的检测为阴性,表明该方法敏感、特异。成功建立了SYBR Green Ⅰ荧光定量PCR检测P1载量的方法,为P1致病机制和机体免疫保护机制的研究提供了技术平台。展开更多
文摘旨在通过RT-PCR扩增猪A群轮状病毒(group A rotavirus,RVA)VP6基因节段,并克隆至pET28a-SUMO,构建原核表达载体pET28a-SUMO-VP6,将该重组载体转化至大肠杆菌BL21(DE3),经IPTG诱导、镍柱亲和层析纯化以获得重组蛋白SUMO-VP6,Western blot验证该重组蛋白能够与小鼠抗轮状病毒血清反应。将该重组蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA筛选阳性杂交瘤细胞,Western blot和间接免疫荧光验证阳性杂交瘤细胞分泌的单克隆抗体与RVA的反应性。经3轮亚克隆最终获得一株能稳定分泌针对VP6蛋白单克隆抗体的阳性杂交瘤细胞株5G9,其分泌的单抗亚类为IgG1,轻链类型为Kappa,制备的小鼠腹水效价可达1∶4096000,能够与不同基因型的RVA反应,同时不与常见的猪腹泻相关病毒反应,为未来猪RVA相关基础研究、临床血清学检测方法的开发奠定了基础。
文摘为探究FILIP1L(filamin A-interacting protein 1-like)蛋白对猪流行性腹泻病毒(PEDV)的影响,以Vero细胞中提取的总RNA反转录的cDNA为模板,PCR扩增出FILIP1L基因,克隆到p3×FLAG-CMV-10真核表达载体上。过表达或沉默FILIP1L基因,通过免疫印迹和定量PCR检测FILIP1L蛋白的表达情况,并研究其抗病毒功能。结果:成功构建3×FLAG-CMV-10-FILIP1L重组质粒,转染后能够正常表达。PEDV感染Vero细胞后,FILIP1L基因的mRNA表达显著上调;过表达FILIP1L基因显著抑制PEDV的复制;下调FILIP1L的表达可显著促进PEDV的复制。综上,FILIP1L蛋白能够抑制PEDV在Vero细胞的复制,研究结果为寻找新的抗PEDV药物靶点和研发新型疫苗提供理论依据。
基金Supported by Natural Science Foundation of Jiangsu Province(BK20131334)Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu Province[CX(13)3069]~~
文摘[Objective] This study aimed to investigate the genetic variation of g E gene of an epidemic pseudorabies virus（PRV） strain and its pathogenicity to piglets. [Method] By serial passage in Vero cells, a PRV strain was isolated from the brain tissues of stillborn fetuses delivered by sows with suspected PRV infection and preliminarily identified by PCR. g E gene of the isolated PRV strain was amplified and sequenced for phylogenetic analysis. In addition, the pathogenicity of the isolated PRV strain to 6-week-old piglets was evaluated. [Result] A PRV strain was successfully isolated and named PRV N5 B strain, which could proliferate in Vero cells and TCID50 of the 15 thgeneration virus liquid reached 10^7.125/0.1 ml. Specific bands could be amplified by PCR. g E gene in the isolated PRV strain was 1 740 bp in length. A phylogenetic tree was constructed based on full-length g E sequences, which showed that PRV N5 B strain and PRV strains isolated since 2012 were clustered into the same independent category and shared 99.7%-100% homology of nucleotide sequences. Compared with related sequences published previously, there were insertions of three consecutive bases at two loci. Animal experiments showed that intranasal inoculation of 6-week-old piglets with 2 ml of PRV N5 B strain（10^6/0.1 ml） led to a mortality rate of 100%. [Conclusion] In this study,genetic variability of g E gene in PRV N5 B isolate and its pathogenicity to piglets were analyzed, which provided a theoretical basis for the development of new vaccines to prevent and control porcine pseudorabies.
文摘该研究旨在建立快速、敏感和特异的检测类猪圆环病毒因子P1的SYBR Green Ⅰ荧光定量PCR法,用于P1的早期诊断。以感染P1的猪血清DNA提取物为模板,采用PCR扩增P1 101 bp的基因片段,将其克隆至pMD18-T载体,重组质粒测序并进行同源性分析;以阳性质粒为模板,建立SYBR Green Ⅰ荧光定量PCR检测方法,并进行敏感性和特异性检测。经测序证实扩增片段属于P1,所建立的SYBR Green Ⅰ荧光定量PCR检测P1的反应在101-108拷贝/μL之间具有良好的线性关系,反应的检出下限为10拷贝/μL,而对猪伪狂犬病病毒、猪细小病毒、猪繁殖与呼吸综合征病毒等的检测为阴性,表明该方法敏感、特异。成功建立了SYBR Green Ⅰ荧光定量PCR检测P1载量的方法,为P1致病机制和机体免疫保护机制的研究提供了技术平台。
