一株白牦牛源牛病毒性腹泻病毒E0基因的原核表达与序列分析
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摘要
【目的】本研究旨在对来源于白牦牛的牛病毒性腹泻病毒(BVDV) GSTZ株的E0基因进行分析,并利用E0基因对GSTZ进行基因分型。【方法】通过提取牛病毒性腹泻病毒(BVDV) RNA,使用RT-PCR技术扩增BVDV E0基因,将其插入到pET-28a中,构建pET-28a-E0原核表达载体,转化BL21(DE3)感受态细胞,筛选阳性克隆,鉴定及测序。IPTG诱导后使用BVDV阳性血清作一抗,经western blot鉴定。根据E0基因的序列进行同源性分析以及构建E0基因系统发育进化树。利用DNAstar预测E0蛋白的抗原表位。【结果】本研究表达的E0蛋白大小与预期结果相符且该蛋白具有良好的抗原性。通过对E0基因序列的同源性分析以及构建E0基因系统发育进化树,说明牛病毒性腹泻病毒GSTZ株基因型可能属于牛病毒性腹泻病毒-1a亚型,也可能是其他基因亚型发生较大突变产生变异。通过预测E0蛋白B细胞的抗原表位主要位于:7-12、22-26、62-63、94-97、101-105、115-119、127-128、138-140、185-187、215-219位氨基酸残基; T细胞潜在优势抗原区域主要位于:10-14、19-31、36-43、52-58、77-86、96-98、137-140、148-150、167-191、209-211位氨基酸残基。【结论】E0蛋白具有良好的抗原性,通过对抗原表位预测以及对GSTZ进行基因分型,为后续基因工程疫苗和BVDV检测试剂盒的开发提供了理论依据。
【Objective】This study was to analyze the E0 gene of bovine viral diarrhea virus(BVDV) GSTZ strain derived from white yak and to genotype the GSTZ strain.【Method】The BVDV structural protein E0 gene was amplified by RT-PCR and inserted into p ET-28 a to construct the p ET-28 a-E0 prokaryotic expression vector by using BL21(DE3) transformation,screening positive clones,identification and sequencing. IPTG induction,western blot identification used BVDV positive serum as primary antibody. According to the sequence of E0 gene homology analysis and construction of phylogenetic tree of E0 gene,DNA star was used to predict the antigenic epitope of E0 protein. 【Result】The results showed that the expressed protein was consistent with the expected. Through the analysis of E0 gene sequence and the construction of E0 gene phylogenetic tree,it is suggested that the genotype of bovine viral diarrhea virus GSTZ strain may belong to bovine viral diarrhea virus-1 a subtype. It is also possible that other subtypes of the gene undergo mutations that produce mutations. We predicted B cell epitope of the E0 protein was mainly located in 7-12,22-26,62-63,94-97,101-105,115-119,127-128,138-140,185-187,215-219 amino acid residues; The potential advantage antigen areas of T cell were mainly located in: 10-14,19-31,36-43,52-58,77-86,96-98,137-140,148-150,167-191,209-211 amino acid residues. 【Conclusion】This study shows that E0 protein has antigenicity. By predicting antigen epitopes and genotyping the GSTZ,it provided the theoretical basis for the development of genetic engineering vaccine and BVDV detection kit.
【Objective】This study was to analyze the E0 gene of bovine viral diarrhea virus(BVDV) GSTZ strain derived from white yak and to genotype the GSTZ strain.【Method】The BVDV structural protein E0 gene was amplified by RT-PCR and inserted into p ET-28 a to construct the p ET-28 a-E0 prokaryotic expression vector by using BL21(DE3) transformation,screening positive clones,identification and sequencing. IPTG induction,western blot identification used BVDV positive serum as primary antibody. According to the sequence of E0 gene homology analysis and construction of phylogenetic tree of E0 gene,DNA star was used to predict the antigenic epitope of E0 protein. 【Result】The results showed that the expressed protein was consistent with the expected. Through the analysis of E0 gene sequence and the construction of E0 gene phylogenetic tree,it is suggested that the genotype of bovine viral diarrhea virus GSTZ strain may belong to bovine viral diarrhea virus-1 a subtype. It is also possible that other subtypes of the gene undergo mutations that produce mutations. We predicted B cell epitope of the E0 protein was mainly located in 7-12,22-26,62-63,94-97,101-105,115-119,127-128,138-140,185-187,215-219 amino acid residues; The potential advantage antigen areas of T cell were mainly located in: 10-14,19-31,36-43,52-58,77-86,96-98,137-140,148-150,167-191,209-211 amino acid residues. 【Conclusion】This study shows that E0 protein has antigenicity. By predicting antigen epitopes and genotyping the GSTZ,it provided the theoretical basis for the development of genetic engineering vaccine and BVDV detection kit.
引文
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[2]Deng Y,Sun C Q,Cao S J,et al. High prevalence of bovine viral diarrhea virus 1 in Chinese swine herds[J]. Veterinary Microbiology,2012,159:490-493.
[3]Goyal S M,Ridpath J F. Bovine Viral Diarrhea Virus:Diagnosis,Management,and Control[J]. Ames:Wiley-Blackwell,2005,272.
[4]Harada T,Tautz N,Thiel H J. E2-P7 Region of the Bovine Viral Diarrhea Virus Polyprotein:Processing and Functional Studies[J]. J Virol,2000,74:9498-9506.
[5]Mztzener P,Magkouras I,Rumenapf T,et al. The viral RNase E(rns)prevents IFN type-I triggering by pestiviral single-and doublestranded RNAs[J]. Virus Res,2009,140:15-23.
[6]Fu Q,Shi H,Shi M,et al.,Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells[J]. Microbial Pathogenesis,2014,76:61-66.
[7]Weiland F,Weiland E,Unger G,et al. Localization of pestiviral envelope proteins E(rns)and E2 at the cell surface and on isolated particles[J]. J Gen Virol,1999,80(5):1157-1165.
[8]Tews B A,Meyers G. The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix[J]. J Biol Chem,2007,282:32730-32741.
[9]Windisch J M,Schneider R,Stark R,et al. RNase of classical swine fever virus:biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies[J]. J Virol,1996,70:352-358.
[10]Vilcek S,Paton D J,Durkovic B,et al. Bovine Viral Diarrhoea Virus Genotype 1 can be Separated into at least Eleven Genetic Groups[J]. Arch Virol,2001,146:99-115.