山羊地方性鼻内肿瘤病毒陕西株序列分析及Gag和Su蛋白多克隆抗体的制备

发布时间：2018-02-21 05:54

本文关键词： ENTV-2 序列分析 Gag蛋白 Su蛋白多克隆抗体　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：羊地方性鼻内肿瘤病毒(Enzootic nasal tumor virus,ENTV)是羊地方性鼻内腺瘤(Enzootic nasal adenocarcinoma,ENA)的病原,可导致鼻甲骨筛骨上皮细胞的致瘤性转化。ENTV-1感染绵羊,ENTV-2感染山羊。因为缺少感染性分子克隆和无法培养病毒,ENTV在ENA发病机理中的作用尚未被完全揭示,GenBank中收录的ENTV全基因组也很少。在中国该病主要流行于内蒙古、湖南、四川、重庆、陕西等多个省市。为了深入研究ENTV,本研究对陕西省疑似ENA的山羊进行诊断,建立了RT-PCR检测方法,对陕西省不同地区的4株ENTV-2进行全基因分段克隆和序列分析,并制备了Gag和Su蛋白多克隆抗体,获得如下研究结果:1.对陕西省4个山羊养殖场发生肿瘤的羊通过临床症状观察,剖检和病理学观察,确定该病为山羊地方性鼻内腺瘤。建立ENTV-2的RT-PCR方法,对临床样品进行检测,阳性率为15.5%。2.通过分段克隆得到陕西省4株ENTV-2的全基因,进行序列比较分析。ENTV-2-Shaanxi1~4全基因和ENTV-2-SC株(登录号:HM104174.1)全基因核苷酸相似度达到98.2~98.7%,和ENTV-2欧洲株(登录号:AY197548.1)相似度达到89.6~89.8%。序列差异部分主要集中在LTR,gag的两个可变区,Orf-x和env的跨膜区(Transmembrane region,Tm)序列。大部分的氨基酸差异存在于Orf-x,ENTV-2-Shaanxi株和ENTV-2-SC株的Orf-x均含有108个氨基酸,而ENTV-2欧洲株含有166个氨基酸。除了Orf-x,还有Gag蛋白的两个可变区域,ENTV-2-Shaanxi1~4株的可变区1区有6个连续的脯氨酸残基。囊膜蛋白的跨膜区,特别是胞质尾区差异也较大,但胞质尾部中的YXXM基序是很保守的,YXXM是PI3K的结合基序。遗传进化分析表明4个Shaanxi株和已发表的2株ENTV-2亲缘关系最近,特别是SC株。3.应用PCR分别扩增gag和su基因并和表达载体pET-28a(+)相连,转化大肠杆菌Rosetta(DE3)株,经IPTG诱导后重组菌成功表达了重组蛋白,将诱导表达的重组蛋白经镍柱亲和层析纯化、复性后免疫小鼠,制备多克隆抗体。Western blot分析试验表明,原核表达纯化的Gag蛋白和Su蛋白能够与制得的鼠抗Gag蛋白和鼠抗Su蛋白多克隆抗体抗体特异性结合,而不与患羊的血清结合。
[Abstract]:Enzootic nasal tumor virus (Enzootic nasal tumor virus) is the pathogen of Enzootic nasal adenocarcinoma Ena. The tumorigenic transformation of ethmoid epithelial cells of nasal turbinate bone. ENTV-1 infected sheep and ENTV-2 infected goats. The role of ENTV-1 in the pathogenesis of ENA has not been fully revealed due to the lack of infectious molecular cloning and the inability to culture the virus. The whole genome of ENTV is also rare. In China, the disease is prevalent in Inner Mongolia. Hunan, Sichuan, Chongqing, Shaanxi and other provinces and cities. In order to deeply study the ENA of goats in Shaanxi Province, a RT-PCR detection method was established for the diagnosis of goats suspected of ENA. Four ENTV-2 strains from different regions of Shaanxi Province were cloned and sequenced, and polyclonal antibodies against Gag and Su proteins were prepared. The following results were obtained: 1. The clinical symptoms of four goat farms in Shaanxi Province were observed. The RT-PCR method of ENTV-2 was established and the positive rate of clinical samples was 15.50.2.The whole genes of 4 strains of ENTV-2 in Shaanxi Province were cloned by segment cloning. The nucleotide similarity between the whole gene and ENTV-2-SC strain (accession number: HM104174.1) was 98.2n 98.7, and that of ENTV-2 European strain (accession No.: AY197548.1) was 89.60.89. 8. The sequence difference was mainly concentrated in the two variable regions of env and LTRgag. The sequence analysis showed that the nucleotide similarity of ENTV-2-Shaanxi1H4 strain (accession number: HM104174.1) was 98.2104174.1, and that of ENTV-2 European strain (accession No.: AY197548.1) was 89.6and 89.8. Most of the amino acid differences were found in the Orf-x of Orf-xENTV-2-Shaanxi strain and ENTV-2-SC strain containing 108 amino acids. In addition to Orf-x, there were two variable regions of Gag protein, ENTV-2-Shaanxi1, which had six continuous proline residues. The transmembrane regions of envelope proteins, especially the cytoplasmic tail region, were also different. However, the YXXM motif in the cytoplasmic tail was conserved as the binding motif of PI3K. Genetic evolution analysis showed that four Shaanxi strains had the closest genetic relationship with the published two ENTV-2 strains. In particular, SC strain 3.The gag and su genes were amplified by PCR and linked to the expression vector pET-28a (), and transformed into E. coli strain Rosetta-DE3. The recombinant protein was successfully expressed by the recombinant strain induced by IPTG, and the recombinant protein was purified by nickel affinity chromatography. After renaturation, mice were immunized with polyclonal antibody. Western blot analysis showed that the purified Gag protein and Su protein expressed in prokaryotic expression could specifically bind to mouse anti-#en2# protein and mouse anti-Su protein polyclonal antibody. Without binding to the serum of the infected sheep.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

【相似文献】