山羊地方性鼻内肿瘤病毒ENTV cDNA文库的构建及功能基因的初步表达
摘要
地方性鼻内肿瘤病是一种以呼吸道鼻内粘膜分泌型上皮细胞发生致瘤性转化为特征的接触性传染病,主要发生于山羊和绵羊,其临床症状主要表现为连续性鼻漏、呼吸困难、眼球突出、颅骨变形、面部肿大、喷嚏、打鼾和极度消瘦。除澳大利亚和新西兰外,所有其他主要养羊国家都有该病的流行,发病率为0.1%-15%,给各国养羊业造成了较大的损失。在国内,·该病主要流行于内蒙古、湖南及四川等主要养羊地区,严重影响了当地山羊种质资源的发展。目前,国内对该病病原学的研究甚少,仅有几例山羊地方性鼻内肿瘤性疾病病理学的相关研究报道。为了深入认识该病,本研究进行了以下几方面的工作：
Ⅰ.山羊地方性鼻内腺瘤病毒ENTV cDNA文库的构建和基因组解析
根据NCBI中收录的ENTV基因组序列设计6对引物,利用RT-PCR技术从山羊地方性鼻内肿瘤自然感染病例的鼻液中的病毒RNA中分段扩增出相应片段,经TA克隆筛选及测序鉴定,成功克隆了重组质粒pMD-ⅰ、pMD-ⅱ、pMD-ⅲ、pMD-ⅳ、pMD-ⅴ、pMD-ⅵ,构建了包含ENTV基因组序列的cDNA文库。序列分析结果显示,相应的gag编码区和env编码区均含有ENTV的独特区,与ENTV-2(登录号：NC_004994)对应序列的相似性分别为88%、90%、93%、88%和92%,而与JSRV及ERVs的差异性较大。序列拼接结果显示,ENTV基因组序列包含5'端非编码区、3'端非编码区和相互重叠的gag-pro-pol-env编码区,基因组全长为7168bp。
Ⅱ.山羊地方性鼻内肿瘤病毒ENTV功能基因的分子克隆及初步表达研究
利用PCR技术从ENTV cNDA文库中成功亚克隆了ENTV gag、env、SU、TM和SD的编码区,构建了pMD-gag、pMD-env、pMD-SU、pMD-TM和pMD-SD的亚克隆,并构建了表达这些编码区的表达质粒(pET-gag、pEGFP-env、pEGFP-SU、pEGFP-TM和pEGFP-SD)。将重组原核表达质粒pET-gag转入宿主菌E.coli Rosetta-gami-TM (DE3) plysS进行表达研究,SDS-PAGE电泳检测到大小约87ku的目的蛋白,为进一步研究gag的功能奠定了基础。重组质粒pEGFP-env、pEGFP-SU、pEGFP-TM、pEGFP-SD转染COS-7哺乳动物细胞,12h后即可观察到荧光,外源基因转录子的RT-PCR检测为阳性,说明重组质粒在启动子的驱动下可以融合荧光蛋白有效表达。
Enzootic nasal tumour (ENT; enzootic nasaladenocarcinoma, ENA) of sheep and goats is a contagious diseases characterized by neoplastic transformation of secretory epithelial cells in the respiratory tract of the mucosal nasal glands. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos, skull deformations, facial swelling, sneezing and snoring respiration and weight loss. The disease occurs naturally in all continents except Australia and New Zealand, the prevalence in sheep was found to be about 0.1%-15%. Epidemiological date indicates that ENT prevalence in the sheep areas such as Inner Mongolia, Hunan and Sichuan, etc, which seriously affected the development of germplasm resources of local goats. There has been little studied in the aetiology and pathophysiology of the ENT in China. The main research contents include three aspects, in order to give further insight regarding the properties of the ENTV.
Ⅰ. cDNA library construction of enzootic nasal tumor virus of SC strain for goats and Characterization of the genome sequence
In order to obtain the cDNA library of enzootic nasal tumor virus of SC strain for goats, the RT-PCR was carried out on nasal fluid from goats with enzootic nasal tumor (ENT), using primers designed referencing to enzootic nasal tumor retrovirus of goats(ENTV-2). The 6 PCR fragments were purified, cloned into the pMD18-T vector and sequenced. The results showed that the genome is 7,168 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. ENTV is highly related to the ENTV-2 sequenced in 2003, Alignment with ENTV-2 gag, pro, pol and env showed 88%、90%、93%、88% and 92% nucleotide homology respectively. ENTV closely related to the retrovirus (ENTV-1) associated with enzootic adenocarcinoma of sheep, and to jaagsiekte retrovirus and endogenous retrovirus. The main sequence differences between these viruses reside in a small regions in gag, the transmembrane (TM) region of env and the U3 LTR.
II. Molecular Cloning and Expression of Functional Gene of enzootic nasal tumor virus of goats strain SC
The PCRs were carried out on the ENTV cDNA libratory, using primers designed from the ORF of gag, env, SU, TM, SD respectively. The PCR fragments 1854bp,1848bp, 1146bp,727bp,150bp were subcloned into pMD18-T simple vector and sequenced. The correct gag ORF was subcoloned into prokaryotic expressing vector pET-32a. The recombinant plasmid pET-gag(1854) was constructed successfully and transformed into E. coli rostta strains. The pET-gag(1854) was induced by IPTG and analysis by SDS-PAGE, the relative molecular weight of expressed product was 87ku, Consistent with the theoretical reasoning. These results will help in the design of a protective vaccine against ENTV infection and the development of ENT. Plasmid DNA (pEGFP-env、pEGFP-SU、pEGFP-TM、pEGFP-SD) were Transfected into COS-7, Fluorescence can be observed after 12h, foreign gene transcripts were detected by RT-PCR, all results showed that foreign gene were fusion expressed effectively in COS-7.
Ⅰ.山羊地方性鼻内腺瘤病毒ENTV cDNA文库的构建和基因组解析
根据NCBI中收录的ENTV基因组序列设计6对引物,利用RT-PCR技术从山羊地方性鼻内肿瘤自然感染病例的鼻液中的病毒RNA中分段扩增出相应片段,经TA克隆筛选及测序鉴定,成功克隆了重组质粒pMD-ⅰ、pMD-ⅱ、pMD-ⅲ、pMD-ⅳ、pMD-ⅴ、pMD-ⅵ,构建了包含ENTV基因组序列的cDNA文库。序列分析结果显示,相应的gag编码区和env编码区均含有ENTV的独特区,与ENTV-2(登录号：NC_004994)对应序列的相似性分别为88%、90%、93%、88%和92%,而与JSRV及ERVs的差异性较大。序列拼接结果显示,ENTV基因组序列包含5'端非编码区、3'端非编码区和相互重叠的gag-pro-pol-env编码区,基因组全长为7168bp。
Ⅱ.山羊地方性鼻内肿瘤病毒ENTV功能基因的分子克隆及初步表达研究
利用PCR技术从ENTV cNDA文库中成功亚克隆了ENTV gag、env、SU、TM和SD的编码区,构建了pMD-gag、pMD-env、pMD-SU、pMD-TM和pMD-SD的亚克隆,并构建了表达这些编码区的表达质粒(pET-gag、pEGFP-env、pEGFP-SU、pEGFP-TM和pEGFP-SD)。将重组原核表达质粒pET-gag转入宿主菌E.coli Rosetta-gami-TM (DE3) plysS进行表达研究,SDS-PAGE电泳检测到大小约87ku的目的蛋白,为进一步研究gag的功能奠定了基础。重组质粒pEGFP-env、pEGFP-SU、pEGFP-TM、pEGFP-SD转染COS-7哺乳动物细胞,12h后即可观察到荧光,外源基因转录子的RT-PCR检测为阳性,说明重组质粒在启动子的驱动下可以融合荧光蛋白有效表达。
Enzootic nasal tumour (ENT; enzootic nasaladenocarcinoma, ENA) of sheep and goats is a contagious diseases characterized by neoplastic transformation of secretory epithelial cells in the respiratory tract of the mucosal nasal glands. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos, skull deformations, facial swelling, sneezing and snoring respiration and weight loss. The disease occurs naturally in all continents except Australia and New Zealand, the prevalence in sheep was found to be about 0.1%-15%. Epidemiological date indicates that ENT prevalence in the sheep areas such as Inner Mongolia, Hunan and Sichuan, etc, which seriously affected the development of germplasm resources of local goats. There has been little studied in the aetiology and pathophysiology of the ENT in China. The main research contents include three aspects, in order to give further insight regarding the properties of the ENTV.
Ⅰ. cDNA library construction of enzootic nasal tumor virus of SC strain for goats and Characterization of the genome sequence
In order to obtain the cDNA library of enzootic nasal tumor virus of SC strain for goats, the RT-PCR was carried out on nasal fluid from goats with enzootic nasal tumor (ENT), using primers designed referencing to enzootic nasal tumor retrovirus of goats(ENTV-2). The 6 PCR fragments were purified, cloned into the pMD18-T vector and sequenced. The results showed that the genome is 7,168 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. ENTV is highly related to the ENTV-2 sequenced in 2003, Alignment with ENTV-2 gag, pro, pol and env showed 88%、90%、93%、88% and 92% nucleotide homology respectively. ENTV closely related to the retrovirus (ENTV-1) associated with enzootic adenocarcinoma of sheep, and to jaagsiekte retrovirus and endogenous retrovirus. The main sequence differences between these viruses reside in a small regions in gag, the transmembrane (TM) region of env and the U3 LTR.
II. Molecular Cloning and Expression of Functional Gene of enzootic nasal tumor virus of goats strain SC
The PCRs were carried out on the ENTV cDNA libratory, using primers designed from the ORF of gag, env, SU, TM, SD respectively. The PCR fragments 1854bp,1848bp, 1146bp,727bp,150bp were subcloned into pMD18-T simple vector and sequenced. The correct gag ORF was subcoloned into prokaryotic expressing vector pET-32a. The recombinant plasmid pET-gag(1854) was constructed successfully and transformed into E. coli rostta strains. The pET-gag(1854) was induced by IPTG and analysis by SDS-PAGE, the relative molecular weight of expressed product was 87ku, Consistent with the theoretical reasoning. These results will help in the design of a protective vaccine against ENTV infection and the development of ENT. Plasmid DNA (pEGFP-env、pEGFP-SU、pEGFP-TM、pEGFP-SD) were Transfected into COS-7, Fluorescence can be observed after 12h, foreign gene transcripts were detected by RT-PCR, all results showed that foreign gene were fusion expressed effectively in COS-7.
引文
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