犬星状病毒（Canineastrovirus，CaAstv）半巢式RT-PCR检测方法的建立及基因序列分析
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摘要
犬星状病毒是无囊膜的单股正链RNA病毒,呈五角或六角的星形,可引起犬呕吐、腹泻、发热等症状,对犬的健康造成威胁。因此建立一种敏感、特异的犬星状病毒的PCR检测方法具有重要意义。本文从犬星状病毒半套式RT-PCR检测方法的建立及初步应用、病毒基因组序列扩增及分析、P1蛋白的表达纯化等方面进行了系统研究,结果如下:
本研究根据GenBank上发表的犬星状病毒ORF2序列5′端保守区设计特异性引物,通过反应条件的优化、特异性实验和敏感性实验,建立了敏感、快速、准确的半套式RT-PCR检测方法。PCR反应的目的片段为ORF2序列5′端保守区的RNA片段,大小为480bp。优化50ul反应体系中,最适引物终浓度为0.24umol/L、最适dNTP浓度为0.4mM、Mg~(2+)浓度为1.5mmol/L、Taq酶浓度为0.05U/ul,退火温度为56℃。与犬细小病毒、犬轮状病毒、犬博卡病毒均无特异性反应。利用建立的半套式RT-PCR检测方法对上海地区321份新鲜采集的犬粪便样品(183份腹泻样品和138份健康样品)进行初步检测。结果显示:腹泻样品中共有22份阳性,阳性率为(12.02%),健康犬粪样中未检测到星状病毒。进化树及同源性分析发现,犬星状病毒中国株与犬星状病毒意大利株分为不同的簇,表明犬星状病毒中国株可能为一种新毒株。试验表明,本研究建立的半套式RT-PCR方法可用于临床粪便样品的检测,具有敏感、快速、准确的特点,该方法的建立为犬星状病毒感染的临床快速诊断提供了科学依据。
根据犬星状病毒GenBank已知序列信息共设计4对引物,结合LA PCRTM和Genome Walking等技术对其中一株病毒的基因序列进行扩增,并对该株病毒的基因序列进行分析。结果显示:本研究扩增出5011bp的基因片段,包括完整的ORF2基因(2475bp)、完整的ORF1b基因(1536bp)以及部分ORF1a基因(1198bp)。将具有抗原表位的ORF2基因5′端保守区RNA片段(P1),克隆到原核表达载体pET-30a上,经酶切、测序鉴定后,将重组质粒转化至大肠杆菌BL21(DE3)中,用终浓度为1mmol.L-1 IPTG诱导,表达产物以包涵体形式存在。超声波裂解菌体后对包涵体进行溶解,再用Ni-NTA柱对重组蛋白进行纯化,并对纯化的蛋白进行复性,为下一步ELISA检测方法的建立奠定了基础。
Canine astroviruses are non-enveloped single-stranded positive sense RNA viruses with a·ve to six point star-like appearance. Generally, astrovirus infections are associated with enteric diseases, with mild to severe signs such as diarrhea, vomiting and abdominal pain. So, it is important to develop a method for canine astrovirus detection. About the semi-nested RT-PCR detection method of canine astrovirus, primers were designed, targeting a relatively conserved region at the 5′-end of ORF2 based on the published genomic sequence of canine astrovirus and this generated a 480-bp amplicon. The best reaction conditions of the method is as follows: the optimal concentration of primers was 0.24umol·L~(-1), the optimal concentration of dNTP was 0.4mM, the optimal concentration of Mg2+ was 1.5mmol·L~(-1), the optimal concentration of Taq enzyme is 0.05U/ul and the optimal annealing temperature is 56℃. Stool specimens were collected from 183 dogs with diarrhea and 138 healthy controls in Shanghai. Canine astrovirus were detected by using this method. 22 (12.02%) specimens from the 183 dogs with diarrhea and none (0%) from the 138 healthy controls were positive for astrovirus. Phylogenetic analysis indicated that the new isolates and the Italy strain were divided into two different clusters and the new isolates may represent a new strain of canine astrovirus. The result showed that the semi-nested RT-PCR assay had the advantage such as high sensibility, strong specificity and this method can be used for the epidemiological investigation and diagnosis of canine astrovirus.
Using a combination of LA PCRTM and Genome Walking, 4 pairs of primers are devised based on those sequences available in GenBank. 5011 nucleotides of the genomic RNA of canine astrovirus were obtained, including the entire ORF2 gene (2475bp), the entire ORF1b gene (1536bp) and partial ORF1a gene (1198bp). Then a relatively conserved region at the 5′-end of ORF2 gene (P1 segment) was amplified by PCR, and inserted into the prokaryotic expression vector pET-30a. After identified by restrict enzyme and sequencing, the constructed recombinant expression plasmid was transformed into the receptive cells of E.coli BL21 (DE3) and induced by IPTG with a finial concentration 1mmol·L~(-1), and the recombinant protein was expressed in the form of inclusion bodies. After the bacterium cell walls were disrupted by ultrasonication, the inclusion bodies of recombinant proteins were dissolved, and then were purified by Ni~(2+) affinity columns following with renaturation. The purified protein may make senses in the coming application in a serological test.
本研究根据GenBank上发表的犬星状病毒ORF2序列5′端保守区设计特异性引物,通过反应条件的优化、特异性实验和敏感性实验,建立了敏感、快速、准确的半套式RT-PCR检测方法。PCR反应的目的片段为ORF2序列5′端保守区的RNA片段,大小为480bp。优化50ul反应体系中,最适引物终浓度为0.24umol/L、最适dNTP浓度为0.4mM、Mg~(2+)浓度为1.5mmol/L、Taq酶浓度为0.05U/ul,退火温度为56℃。与犬细小病毒、犬轮状病毒、犬博卡病毒均无特异性反应。利用建立的半套式RT-PCR检测方法对上海地区321份新鲜采集的犬粪便样品(183份腹泻样品和138份健康样品)进行初步检测。结果显示:腹泻样品中共有22份阳性,阳性率为(12.02%),健康犬粪样中未检测到星状病毒。进化树及同源性分析发现,犬星状病毒中国株与犬星状病毒意大利株分为不同的簇,表明犬星状病毒中国株可能为一种新毒株。试验表明,本研究建立的半套式RT-PCR方法可用于临床粪便样品的检测,具有敏感、快速、准确的特点,该方法的建立为犬星状病毒感染的临床快速诊断提供了科学依据。
根据犬星状病毒GenBank已知序列信息共设计4对引物,结合LA PCRTM和Genome Walking等技术对其中一株病毒的基因序列进行扩增,并对该株病毒的基因序列进行分析。结果显示:本研究扩增出5011bp的基因片段,包括完整的ORF2基因(2475bp)、完整的ORF1b基因(1536bp)以及部分ORF1a基因(1198bp)。将具有抗原表位的ORF2基因5′端保守区RNA片段(P1),克隆到原核表达载体pET-30a上,经酶切、测序鉴定后,将重组质粒转化至大肠杆菌BL21(DE3)中,用终浓度为1mmol.L-1 IPTG诱导,表达产物以包涵体形式存在。超声波裂解菌体后对包涵体进行溶解,再用Ni-NTA柱对重组蛋白进行纯化,并对纯化的蛋白进行复性,为下一步ELISA检测方法的建立奠定了基础。
Canine astroviruses are non-enveloped single-stranded positive sense RNA viruses with a·ve to six point star-like appearance. Generally, astrovirus infections are associated with enteric diseases, with mild to severe signs such as diarrhea, vomiting and abdominal pain. So, it is important to develop a method for canine astrovirus detection. About the semi-nested RT-PCR detection method of canine astrovirus, primers were designed, targeting a relatively conserved region at the 5′-end of ORF2 based on the published genomic sequence of canine astrovirus and this generated a 480-bp amplicon. The best reaction conditions of the method is as follows: the optimal concentration of primers was 0.24umol·L~(-1), the optimal concentration of dNTP was 0.4mM, the optimal concentration of Mg2+ was 1.5mmol·L~(-1), the optimal concentration of Taq enzyme is 0.05U/ul and the optimal annealing temperature is 56℃. Stool specimens were collected from 183 dogs with diarrhea and 138 healthy controls in Shanghai. Canine astrovirus were detected by using this method. 22 (12.02%) specimens from the 183 dogs with diarrhea and none (0%) from the 138 healthy controls were positive for astrovirus. Phylogenetic analysis indicated that the new isolates and the Italy strain were divided into two different clusters and the new isolates may represent a new strain of canine astrovirus. The result showed that the semi-nested RT-PCR assay had the advantage such as high sensibility, strong specificity and this method can be used for the epidemiological investigation and diagnosis of canine astrovirus.
Using a combination of LA PCRTM and Genome Walking, 4 pairs of primers are devised based on those sequences available in GenBank. 5011 nucleotides of the genomic RNA of canine astrovirus were obtained, including the entire ORF2 gene (2475bp), the entire ORF1b gene (1536bp) and partial ORF1a gene (1198bp). Then a relatively conserved region at the 5′-end of ORF2 gene (P1 segment) was amplified by PCR, and inserted into the prokaryotic expression vector pET-30a. After identified by restrict enzyme and sequencing, the constructed recombinant expression plasmid was transformed into the receptive cells of E.coli BL21 (DE3) and induced by IPTG with a finial concentration 1mmol·L~(-1), and the recombinant protein was expressed in the form of inclusion bodies. After the bacterium cell walls were disrupted by ultrasonication, the inclusion bodies of recombinant proteins were dissolved, and then were purified by Ni~(2+) affinity columns following with renaturation. The purified protein may make senses in the coming application in a serological test.
引文
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