猪繁殖与呼吸综合征病毒河北地方株ORF7基因的原核表达及ELISA方法的建立
发布时间：2019-01-11 12:22
【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种母猪繁殖障碍和仔猪呼吸道症状的传染病,又称猪蓝耳病。PRRS已成为当前困扰中国养猪业健康发展的主要烈性传染病之一。本课题旨在从河北省本地猪场分离毒株,通过与Genbank上发表的核苷酸和氨基酸序列进行对比,了解河北PRRSV的分子流行病学特点。以本地分离株为基础,克隆表达编码PRRS病毒粒子中含量最多、免疫原性最强蛋白的ORF7基因,构建成熟的原核表达系统,并将表达的N蛋白作为包被抗原建立间接ELISA方法。本研究从河北省多地送检的疑似PRRS阳性病料进行RT-PCR鉴定,将确诊的阳性病料接种Marc-145细胞,然后连续传代。其中河北新乐市送检病料接种Marc-145细胞后经过5代盲传后,出现典型细胞病变(CPE),初步分离到一株病毒,暂命名为PRRSV HB-XL株。进一步对其ORF5与NSP2基因克隆测序,经同源性比较及系统进化树等分析,表明该毒株ORF5与NSP2基因与国内流行的缺失变异株遗传关系较近,与国内外经典美洲型毒株及基因重组毒株QYYZ遗传关系较远且与欧洲型LV毒株处于不同分支;ORF5基因编码的200个氨基酸的第13位、151位均为具有强毒特性的精氨酸(R),第137位为具有野毒特性的丝氨酸(S);与经典美洲株相比,该毒株NSP2基因分别发生3个碱基和87个碱基的不连续缺失。说明HB-XL株属于美洲型PRRSV并存在不断变异的趋势。设计1对添加酶切位点的特异性引物扩增PRRSV HB-XL株的ORF7全基因,并与原核表达载体p ET-32a连接,构建ORF7基因的原核表达系统p ET-32a-N;将重组质粒转化入BL21感受态细胞,经IPTG的诱导表达,SDS-PAGE可检测到分子质量约为34.0k Da的目的蛋白;经Western-blotting分析,表明该重组蛋白具有良好的免疫原性;并对表达条件进行了优化,结果表明在诱导时间为4h,IPTG浓度1.5m M,诱导温度为37℃的条件下目的蛋白表达量最大。将表达纯化后的N蛋白作为包被抗原,按一定的稀释度倍比稀释。采集PRRSV阳性猪血清,按一定稀释度倍比稀释。测定各步骤反应的条件,成功建立ELISA方法。重组抗原包被浓度为2.23μg/ml,37℃1h或者4℃过夜;封闭液为2%的明胶,37℃封闭1 h;血清1:80稀释,37℃作用1h;酶标二抗1:1000稀释,37℃作用40min;底物(TMB)室温显色15min;若检测的OD4500.363则为阳性,若检测的OD4500.34则为阴性。该方法与商业化的美国IDEXX PRRSV抗体检测试剂盒的符合率为91.6%,其中相对敏感性为92.3%,相对特异性为90.4%,结果无显著差异。说明本研究建立的间接ELISA方法具有良好的特异性、敏感性,有很好的临床应用前景。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus,PRRSV) in sows and respiratory symptoms in piglets. PRRS has become one of the major infectious diseases that haunt the healthy development of pig industry in China. The purpose of this study was to isolate the strains from local pig farms in Hebei Province and to compare the nucleotide and amino acid sequences published on Genbank to understand the molecular epidemiological characteristics of PRRSV in Hebei Province. Based on the native isolates, the ORF7 gene encoding the largest amount of PRRS virus particles and the strongest immunogenicity protein was cloned and expressed. The mature prokaryotic expression system was constructed. The expressed N protein was used as the coated antigen to establish an indirect ELISA method. In this study, the suspected PRRS positive venereal disease materials from Hebei Province were identified by RT-PCR. The positive venereal materials were inoculated with Marc-145 cells and then subcultured continuously. After inoculation of Marc-145 cells in Xinle City, Hebei Province, after 5 generations of blind transmission, a virus strain was initially isolated from typical cytopathic (CPE), which was tentatively named PRRSV HB-XL strain. Further cloning and sequencing of its ORF5 and NSP2 genes, homology comparison and phylogenetic tree analysis showed that the ORF5 and NSP2 genes of the virus strain had close genetic relationship with the prevalent mutant strains in China. The genetic relationship between QYYZ and the classical American strain and recombinant strain was far away and was different from that of the European type LV strain. ORF5 gene encodes the 13th of 200 amino acids, 151 of which are arginine (R), with strong toxicity, 137 of which are serine (S); with wild toxicity. Compared with the classical American strain, the NSP2 gene of this strain showed discontinuous deletion of 3 bases and 87 bases, respectively. The results showed that the HB-XL strain belonged to American type PRRSV and had a trend of continuous variation. The whole ORF7 gene of PRRSV HB-XL strain was amplified by a pair of specific primers with restriction endonuclease site, and ligated with prokaryotic expression vector p ET-32a. A prokaryotic expression system of ORF7 gene, p ET-32a-N;, was constructed. The recombinant plasmid was transformed into BL21 competent cells and expressed by IPTG. The target protein with molecular weight of about 34.0k Da could be detected by SDS-PAGE, and the result of Western-blotting analysis showed that the recombinant protein had good immunogenicity. The expression conditions were optimized. The results showed that the maximum expression of the target protein was obtained when the induction time was 4 h, the concentration of IPTG was 1.5 mm, and the induction temperature was 37 鈩，
本文编号：2407125
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus,PRRSV) in sows and respiratory symptoms in piglets. PRRS has become one of the major infectious diseases that haunt the healthy development of pig industry in China. The purpose of this study was to isolate the strains from local pig farms in Hebei Province and to compare the nucleotide and amino acid sequences published on Genbank to understand the molecular epidemiological characteristics of PRRSV in Hebei Province. Based on the native isolates, the ORF7 gene encoding the largest amount of PRRS virus particles and the strongest immunogenicity protein was cloned and expressed. The mature prokaryotic expression system was constructed. The expressed N protein was used as the coated antigen to establish an indirect ELISA method. In this study, the suspected PRRS positive venereal disease materials from Hebei Province were identified by RT-PCR. The positive venereal materials were inoculated with Marc-145 cells and then subcultured continuously. After inoculation of Marc-145 cells in Xinle City, Hebei Province, after 5 generations of blind transmission, a virus strain was initially isolated from typical cytopathic (CPE), which was tentatively named PRRSV HB-XL strain. Further cloning and sequencing of its ORF5 and NSP2 genes, homology comparison and phylogenetic tree analysis showed that the ORF5 and NSP2 genes of the virus strain had close genetic relationship with the prevalent mutant strains in China. The genetic relationship between QYYZ and the classical American strain and recombinant strain was far away and was different from that of the European type LV strain. ORF5 gene encodes the 13th of 200 amino acids, 151 of which are arginine (R), with strong toxicity, 137 of which are serine (S); with wild toxicity. Compared with the classical American strain, the NSP2 gene of this strain showed discontinuous deletion of 3 bases and 87 bases, respectively. The results showed that the HB-XL strain belonged to American type PRRSV and had a trend of continuous variation. The whole ORF7 gene of PRRSV HB-XL strain was amplified by a pair of specific primers with restriction endonuclease site, and ligated with prokaryotic expression vector p ET-32a. A prokaryotic expression system of ORF7 gene, p ET-32a-N;, was constructed. The recombinant plasmid was transformed into BL21 competent cells and expressed by IPTG. The target protein with molecular weight of about 34.0k Da could be detected by SDS-PAGE, and the result of Western-blotting analysis showed that the recombinant protein had good immunogenicity. The expression conditions were optimized. The results showed that the maximum expression of the target protein was obtained when the induction time was 4 h, the concentration of IPTG was 1.5 mm, and the induction temperature was 37 鈩，
本文编号：2407125
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