我国部分地区PRRSV的分子流行病学分析与NADC30-like株GP5蛋白的原核表达
发布时间：2019-03-02 20:43
【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是一种广泛传播且致病性较强的病毒性疾病,其特征是母猪繁殖障碍,不同年龄段猪群表现出呼吸系统症状,对全球养猪业造成严重的经济损失。由于该病毒具有很高的变异性,为了解我国近两年内PRRS的流行状况及变异特点,同时对流行毒株进一步探索。本研究对我国部分地区PRRSV分子流行病学进行分析,完成了流行毒株的毒株分离及GP5蛋白的原核表达,主要内容如下:1.对2015~2016年收集自我国23个省份的发病猪场PRRS疑似病料进行PRRSV病原检测,并对部分具有代表性的阳性病料进行ORF5基因的扩增测序,得到了177个PRRSV ORF5基因序列。结果显示,在3078份疑似病料中,共检测出阳性样本889份,阳性率为28.88%;对得到的ORF5基因序列进行分析,结果显示,177个毒株均属于美洲型毒株,其中90株与我国的HP-PRRSV亚群代表株JXA1高度同源,34株与NADC30高度同源。与2015年相比,2016年我国PRRSV的流行仍以HP-PRRSV为主要流行毒株,NADC30-like毒株在流行毒株中的占比上升了12.04%。同时,PRRSV的流行不具有明显的地域特性。对ORF5基因的推导氨基酸序列进行分析,结果显示部分NADC30-like毒株在N34位糖基化位点存在缺失,提示有必要继续进行PRRSV的分子流行病学监测,为下一步的防控提供依据。2.将PRRSV阳性组织病料进研磨后接种到Marc-145细胞中,连续传代进行病毒分离,在传至第3~4代时出现明显细胞病变。继续传代的同时,对其进行RT-PCR鉴定和测定病毒效价,通过测序验证后确定分离到的3株病毒均为PRRS病毒,分别命名为JX1、JX2、SD1。毒株序列分析后显示3株毒株均属于NADC30-like毒株,对第5代病毒液的病毒效价进行测定,结果显示病毒含量分别为10-6TCID50/0.1m L、10-5.33TCID50/0.1mL、10-5.67TCID50/0.1mL。3.对分离的一株PRRSV流行毒株(JX1)的GP5基因进行扩增,以GP5基因的重组质粒载体为模板分别扩增两条片段,通过融合PCR得到缺失GP5信号肽和跨膜区的基因片段(dORF5),将其克隆到表达载体pGEX-6P-1上。将原核表达载体pGEX-6P-1转化大肠杆菌BL21,用浓度为1 mmol/L的IPTG在37℃条件下诱导。SDS-PAGE电泳检测,所表达蛋白的分子量约为38 kDa,对诱导时间进行优化后发现在5 h时表达量达到最大。对蛋白可溶性进行鉴定显示所表达的部分蛋白具有良好的可溶性。Western Blot结果表明表达出的蛋白与PRRSV阳性血清发生发应,表明所表达的蛋白具有良好的反应原性。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is a widely spread and highly pathogenic viral disease characterized by reproductive disorders in sows and respiratory symptoms in pigs of different ages. It has caused serious economic losses to the global pig industry. Because of the high variability of the virus, in order to understand the epidemic situation and variation characteristics of PRRS in China in the last two years, and to further explore the epidemic strains. In this study, the molecular epidemiology of PRRSV in some areas of China was analyzed, and the isolates of the epidemic strains and the prokaryotic expression of GP5 protein were completed. The main contents are as follows: 1. The suspected PRRS samples collected from 23 provinces of China in 2015 / 2016 were detected for PRRSV pathogen, and some representative positive samples were amplified by ORF5 gene, and 177 PRRSV ORF5 gene sequences were obtained. The results showed that out of 3078 suspected samples, 889 positive samples were detected, the positive rate was 28.88%. The results showed that all strains belonged to American type, of which 90 strains were highly homologous to the representative strain JXA1 of HP-PRRSV subgroup in China and 34 strains were highly homologous to NADC30. The sequence of ORF5 gene was analyzed. Compared with 2015, the prevalence of PRRSV in China in 2016 was still dominated by HP-PRRSV, and the proportion of NADC30-like strains in the epidemic strains increased by 12.04%. At the same time, the popularity of PRRSV has no obvious geographical characteristics. The deduced amino acid sequence of ORF5 gene was analyzed and the results showed that some of the NADC30-like strains were deleted at the glycosylation site at N34 site, suggesting that it is necessary to continue the molecular epidemiological surveillance of PRRSV in order to provide the basis for the next step of prevention and control. 2. PRRSV positive tissue material was inoculated into Marc-145 cells after grinding, and virus isolation was carried out continuously. Obvious cytopathic effects were observed at passage 3 ~ 4. At the same time, RT-PCR was used to identify the virus and determine the titer of the virus. After sequencing, it was confirmed that the three strains of the virus were PRRS virus, named JX1,JX2,SD1., respectively. Sequence analysis showed that the three strains belonged to NADC30-like strain. The titer of the fifth generation virus was determined. The results showed that the virus content was 10-6TCID50/0.1m / L, 10 脳 5. 33 TCID _ (50) ~ (0.1 mL) and 10 ~ (- 5) TCID _ (50) 脳 10 ~ (- 1) mL, respectively. 10 / 5.67 TCID50 / 0.1mL.3. The GP5 gene of a PRRSV epidemic strain (JX1) was amplified. The recombinant plasmid vector of GP5 gene was used as template to amplify two fragments respectively. The GP5 signal peptide and transmembrane region gene fragment (dORF5) were obtained by fusion of PCR. It was cloned into the expression vector pGEX-6P-1. The prokaryotic expression vector pGEX-6P-1 was transformed into E. coli BL21, and induced by IPTG with concentration of 1 mmol/L at 37 鈩，
本文编号：2433454
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is a widely spread and highly pathogenic viral disease characterized by reproductive disorders in sows and respiratory symptoms in pigs of different ages. It has caused serious economic losses to the global pig industry. Because of the high variability of the virus, in order to understand the epidemic situation and variation characteristics of PRRS in China in the last two years, and to further explore the epidemic strains. In this study, the molecular epidemiology of PRRSV in some areas of China was analyzed, and the isolates of the epidemic strains and the prokaryotic expression of GP5 protein were completed. The main contents are as follows: 1. The suspected PRRS samples collected from 23 provinces of China in 2015 / 2016 were detected for PRRSV pathogen, and some representative positive samples were amplified by ORF5 gene, and 177 PRRSV ORF5 gene sequences were obtained. The results showed that out of 3078 suspected samples, 889 positive samples were detected, the positive rate was 28.88%. The results showed that all strains belonged to American type, of which 90 strains were highly homologous to the representative strain JXA1 of HP-PRRSV subgroup in China and 34 strains were highly homologous to NADC30. The sequence of ORF5 gene was analyzed. Compared with 2015, the prevalence of PRRSV in China in 2016 was still dominated by HP-PRRSV, and the proportion of NADC30-like strains in the epidemic strains increased by 12.04%. At the same time, the popularity of PRRSV has no obvious geographical characteristics. The deduced amino acid sequence of ORF5 gene was analyzed and the results showed that some of the NADC30-like strains were deleted at the glycosylation site at N34 site, suggesting that it is necessary to continue the molecular epidemiological surveillance of PRRSV in order to provide the basis for the next step of prevention and control. 2. PRRSV positive tissue material was inoculated into Marc-145 cells after grinding, and virus isolation was carried out continuously. Obvious cytopathic effects were observed at passage 3 ~ 4. At the same time, RT-PCR was used to identify the virus and determine the titer of the virus. After sequencing, it was confirmed that the three strains of the virus were PRRS virus, named JX1,JX2,SD1., respectively. Sequence analysis showed that the three strains belonged to NADC30-like strain. The titer of the fifth generation virus was determined. The results showed that the virus content was 10-6TCID50/0.1m / L, 10 脳 5. 33 TCID _ (50) ~ (0.1 mL) and 10 ~ (- 5) TCID _ (50) 脳 10 ~ (- 1) mL, respectively. 10 / 5.67 TCID50 / 0.1mL.3. The GP5 gene of a PRRSV epidemic strain (JX1) was amplified. The recombinant plasmid vector of GP5 gene was used as template to amplify two fragments respectively. The GP5 signal peptide and transmembrane region gene fragment (dORF5) were obtained by fusion of PCR. It was cloned into the expression vector pGEX-6P-1. The prokaryotic expression vector pGEX-6P-1 was transformed into E. coli BL21, and induced by IPTG with concentration of 1 mmol/L at 37 鈩，
本文编号：2433454
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