我國部分地區(qū)PRRSV的分子流行病學(xué)分析與NADC30-like株GP5蛋白的原核表達(dá)
發(fā)布時(shí)間：2019-03-02 20:43
【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)是一種廣泛傳播且致病性較強(qiáng)的病毒性疾病,其特征是母豬繁殖障礙,不同年齡段豬群表現(xiàn)出呼吸系統(tǒng)癥狀,對全球養(yǎng)豬業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。由于該病毒具有很高的變異性,為了解我國近兩年內(nèi)PRRS的流行狀況及變異特點(diǎn),同時(shí)對流行毒株進(jìn)一步探索。本研究對我國部分地區(qū)PRRSV分子流行病學(xué)進(jìn)行分析,完成了流行毒株的毒株分離及GP5蛋白的原核表達(dá),主要內(nèi)容如下:1.對2015~2016年收集自我國23個(gè)省份的發(fā)病豬場PRRS疑似病料進(jìn)行PRRSV病原檢測,并對部分具有代表性的陽性病料進(jìn)行ORF5基因的擴(kuò)增測序,得到了177個(gè)PRRSV ORF5基因序列。結(jié)果顯示,在3078份疑似病料中,共檢測出陽性樣本889份,陽性率為28.88%;對得到的ORF5基因序列進(jìn)行分析,結(jié)果顯示,177個(gè)毒株均屬于美洲型毒株,其中90株與我國的HP-PRRSV亞群代表株JXA1高度同源,34株與NADC30高度同源。與2015年相比,2016年我國PRRSV的流行仍以HP-PRRSV為主要流行毒株,NADC30-like毒株在流行毒株中的占比上升了12.04%。同時(shí),PRRSV的流行不具有明顯的地域特性。對ORF5基因的推導(dǎo)氨基酸序列進(jìn)行分析,結(jié)果顯示部分NADC30-like毒株在N34位糖基化位點(diǎn)存在缺失,提示有必要繼續(xù)進(jìn)行PRRSV的分子流行病學(xué)監(jiān)測,為下一步的防控提供依據(jù)。2.將PRRSV陽性組織病料進(jìn)研磨后接種到Marc-145細(xì)胞中,連續(xù)傳代進(jìn)行病毒分離,在傳至第3~4代時(shí)出現(xiàn)明顯細(xì)胞病變。繼續(xù)傳代的同時(shí),對其進(jìn)行RT-PCR鑒定和測定病毒效價(jià),通過測序驗(yàn)證后確定分離到的3株病毒均為PRRS病毒,分別命名為JX1、JX2、SD1。毒株序列分析后顯示3株毒株均屬于NADC30-like毒株,對第5代病毒液的病毒效價(jià)進(jìn)行測定,結(jié)果顯示病毒含量分別為10-6TCID50/0.1m L、10-5.33TCID50/0.1mL、10-5.67TCID50/0.1mL。3.對分離的一株P(guān)RRSV流行毒株(JX1)的GP5基因進(jìn)行擴(kuò)增,以GP5基因的重組質(zhì)粒載體為模板分別擴(kuò)增兩條片段,通過融合PCR得到缺失GP5信號肽和跨膜區(qū)的基因片段(dORF5),將其克隆到表達(dá)載體pGEX-6P-1上。將原核表達(dá)載體pGEX-6P-1轉(zhuǎn)化大腸桿菌BL21,用濃度為1 mmol/L的IPTG在37℃條件下誘導(dǎo)。SDS-PAGE電泳檢測,所表達(dá)蛋白的分子量約為38 kDa,對誘導(dǎo)時(shí)間進(jìn)行優(yōu)化后發(fā)現(xiàn)在5 h時(shí)表達(dá)量達(dá)到最大。對蛋白可溶性進(jìn)行鑒定顯示所表達(dá)的部分蛋白具有良好的可溶性。Western Blot結(jié)果表明表達(dá)出的蛋白與PRRSV陽性血清發(fā)生發(fā)應(yīng),表明所表達(dá)的蛋白具有良好的反應(yīng)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，
本文編號：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，
本文編號：2433454
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