豬繁殖與呼吸綜合征病毒河北地方株ORF7基因的原核表達(dá)及ELISA方法的建立
發(fā)布時(shí)間：2019-01-11 12:22
【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的一種母豬繁殖障礙和仔豬呼吸道癥狀的傳染病,又稱(chēng)豬藍(lán)耳病。PRRS已成為當(dāng)前困擾中國(guó)養(yǎng)豬業(yè)健康發(fā)展的主要烈性傳染病之一。本課題旨在從河北省本地豬場(chǎng)分離毒株,通過(guò)與Genbank上發(fā)表的核苷酸和氨基酸序列進(jìn)行對(duì)比,了解河北P(pán)RRSV的分子流行病學(xué)特點(diǎn)。以本地分離株為基礎(chǔ),克隆表達(dá)編碼PRRS病毒粒子中含量最多、免疫原性最強(qiáng)蛋白的ORF7基因,構(gòu)建成熟的原核表達(dá)系統(tǒng),并將表達(dá)的N蛋白作為包被抗原建立間接ELISA方法。本研究從河北省多地送檢的疑似PRRS陽(yáng)性病料進(jìn)行RT-PCR鑒定,將確診的陽(yáng)性病料接種Marc-145細(xì)胞,然后連續(xù)傳代。其中河北新樂(lè)市送檢病料接種Marc-145細(xì)胞后經(jīng)過(guò)5代盲傳后,出現(xiàn)典型細(xì)胞病變(CPE),初步分離到一株病毒,暫命名為PRRSV HB-XL株。進(jìn)一步對(duì)其ORF5與NSP2基因克隆測(cè)序,經(jīng)同源性比較及系統(tǒng)進(jìn)化樹(shù)等分析,表明該毒株ORF5與NSP2基因與國(guó)內(nèi)流行的缺失變異株遺傳關(guān)系較近,與國(guó)內(nèi)外經(jīng)典美洲型毒株及基因重組毒株QYYZ遺傳關(guān)系較遠(yuǎn)且與歐洲型LV毒株處于不同分支;ORF5基因編碼的200個(gè)氨基酸的第13位、151位均為具有強(qiáng)毒特性的精氨酸(R),第137位為具有野毒特性的絲氨酸(S);與經(jīng)典美洲株相比,該毒株NSP2基因分別發(fā)生3個(gè)堿基和87個(gè)堿基的不連續(xù)缺失。說(shuō)明HB-XL株屬于美洲型PRRSV并存在不斷變異的趨勢(shì)。設(shè)計(jì)1對(duì)添加酶切位點(diǎn)的特異性引物擴(kuò)增PRRSV HB-XL株的ORF7全基因,并與原核表達(dá)載體p ET-32a連接,構(gòu)建ORF7基因的原核表達(dá)系統(tǒng)p ET-32a-N;將重組質(zhì)粒轉(zhuǎn)化入BL21感受態(tài)細(xì)胞,經(jīng)IPTG的誘導(dǎo)表達(dá),SDS-PAGE可檢測(cè)到分子質(zhì)量約為34.0k Da的目的蛋白;經(jīng)Western-blotting分析,表明該重組蛋白具有良好的免疫原性;并對(duì)表達(dá)條件進(jìn)行了優(yōu)化,結(jié)果表明在誘導(dǎo)時(shí)間為4h,IPTG濃度1.5m M,誘導(dǎo)溫度為37℃的條件下目的蛋白表達(dá)量最大。將表達(dá)純化后的N蛋白作為包被抗原,按一定的稀釋度倍比稀釋。采集PRRSV陽(yáng)性豬血清,按一定稀釋度倍比稀釋。測(cè)定各步驟反應(yīng)的條件,成功建立ELISA方法。重組抗原包被濃度為2.23μg/ml,37℃1h或者4℃過(guò)夜;封閉液為2%的明膠,37℃封閉1 h;血清1:80稀釋,37℃作用1h;酶標(biāo)二抗1:1000稀釋,37℃作用40min;底物(TMB)室溫顯色15min;若檢測(cè)的OD4500.363則為陽(yáng)性,若檢測(cè)的OD4500.34則為陰性。該方法與商業(yè)化的美國(guó)IDEXX PRRSV抗體檢測(cè)試劑盒的符合率為91.6%,其中相對(duì)敏感性為92.3%,相對(duì)特異性為90.4%,結(jié)果無(wú)顯著差異。說(shuō)明本研究建立的間接ELISA方法具有良好的特異性、敏感性,有很好的臨床應(yīng)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，
本文編號(hào)：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，
本文編號(hào)：2407125
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