本文關(guān)鍵詞： ENTV-2 序列分析 Gag蛋白 Su蛋白 多克隆抗體　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：羊地方性鼻內(nèi)腫瘤病毒(Enzootic nasal tumor virus,ENTV)是羊地方性鼻內(nèi)腺瘤(Enzootic nasal adenocarcinoma,ENA)的病原,可導(dǎo)致鼻甲骨篩骨上皮細(xì)胞的致瘤性轉(zhuǎn)化。ENTV-1感染綿羊,ENTV-2感染山羊。因?yàn)槿鄙俑腥拘苑肿涌寺『蜔o(wú)法培養(yǎng)病毒,ENTV在ENA發(fā)病機(jī)理中的作用尚未被完全揭示,GenBank中收錄的ENTV全基因組也很少。在中國(guó)該病主要流行于內(nèi)蒙古、湖南、四川、重慶、陜西等多個(gè)省市。為了深入研究ENTV,本研究對(duì)陜西省疑似ENA的山羊進(jìn)行診斷,建立了RT-PCR檢測(cè)方法,對(duì)陜西省不同地區(qū)的4株ENTV-2進(jìn)行全基因分段克隆和序列分析,并制備了Gag和Su蛋白多克隆抗體,獲得如下研究結(jié)果:1.對(duì)陜西省4個(gè)山羊養(yǎng)殖場(chǎng)發(fā)生腫瘤的羊通過(guò)臨床癥狀觀察,剖檢和病理學(xué)觀察,確定該病為山羊地方性鼻內(nèi)腺瘤。建立ENTV-2的RT-PCR方法,對(duì)臨床樣品進(jìn)行檢測(cè),陽(yáng)性率為15.5%。2.通過(guò)分段克隆得到陜西省4株ENTV-2的全基因,進(jìn)行序列比較分析。ENTV-2-Shaanxi1~4全基因和ENTV-2-SC株(登錄號(hào):HM104174.1)全基因核苷酸相似度達(dá)到98.2~98.7%,和ENTV-2歐洲株(登錄號(hào):AY197548.1)相似度達(dá)到89.6~89.8%。序列差異部分主要集中在LTR,gag的兩個(gè)可變區(qū),Orf-x和env的跨膜區(qū)(Transmembrane region,Tm)序列。大部分的氨基酸差異存在于Orf-x,ENTV-2-Shaanxi株和ENTV-2-SC株的Orf-x均含有108個(gè)氨基酸,而ENTV-2歐洲株含有166個(gè)氨基酸。除了Orf-x,還有Gag蛋白的兩個(gè)可變區(qū)域,ENTV-2-Shaanxi1~4株的可變區(qū)1區(qū)有6個(gè)連續(xù)的脯氨酸殘基。囊膜蛋白的跨膜區(qū),特別是胞質(zhì)尾區(qū)差異也較大,但胞質(zhì)尾部中的YXXM基序是很保守的,YXXM是PI3K的結(jié)合基序。遺傳進(jìn)化分析表明4個(gè)Shaanxi株和已發(fā)表的2株ENTV-2親緣關(guān)系最近,特別是SC株。3.應(yīng)用PCR分別擴(kuò)增gag和su基因并和表達(dá)載體pET-28a(+)相連,轉(zhuǎn)化大腸桿菌Rosetta(DE3)株,經(jīng)IPTG誘導(dǎo)后重組菌成功表達(dá)了重組蛋白,將誘導(dǎo)表達(dá)的重組蛋白經(jīng)鎳柱親和層析純化、復(fù)性后免疫小鼠,制備多克隆抗體。Western blot分析試驗(yàn)表明,原核表達(dá)純化的Gag蛋白和Su蛋白能夠與制得的鼠抗Gag蛋白和鼠抗Su蛋白多克隆抗體抗體特異性結(jié)合,而不與患羊的血清結(jié)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊威,張寶山,劉永剛,劉勝旺,孔憲剛,曹殿軍,劉相冬,盧景良,劉寶全,劉建華;馬傳染性貧血病毒驢白細(xì)胞弱毒疫苗gag基因的序列測(cè)定及分析[J];病毒學(xué)報(bào);2000年04期

2 韓保光,孟莉,鄒民吉,凌世淦,宋曉國(guó),趙春文,段聚寶,王嘉璽,馬賢凱;人類(lèi)免疫缺陷病毒Ⅰ型Gag前體蛋白片段在大腸桿菌中的表達(dá)、純化及鑒定[J];生物技術(shù)通訊;1996年02期

3 Yoko M,石偉光;Ⅰ型人類(lèi)免疫缺陷病毒Gag病毒樣顆粒的體外組裝[J];國(guó)外醫(yī)學(xué).病毒學(xué)分冊(cè);2001年01期

4 張錦霞,金寧一,方厚華,羅坤,郭焱,邵一鳴,殷震;Ⅰ型人免疫缺陷病毒(HIV-1)中國(guó)流行株B亞型Gag和Env蛋白在大腸桿菌中的表達(dá)[J];中國(guó)獸醫(yī)學(xué)報(bào);2001年03期

5 田梅,金寧一,王秀清,郭志儒,李體遠(yuǎn),李宗株,方厚華,范洪學(xué),殷震;人Ⅱ型免疫缺陷病毒gag基因在大腸桿菌中的表達(dá)[J];中國(guó)免疫學(xué)雜志;1999年06期

6 耿慶華;王曉鈞;相文華;沈榮顯;;馬傳染性貧血病毒阿根廷流行毒株前病毒gag和gp90基因在馬體內(nèi)的變異分析[J];畜牧獸醫(yī)學(xué)報(bào);2006年03期

7 張洪濤,邵一鳴,肖瑤,潘品良,季陽(yáng);HIV-1型中國(guó)株E、B亞型gag基因的克隆與表達(dá)[J];中華實(shí)驗(yàn)和臨床病毒學(xué)雜志;2000年01期

8 ;HIV-1中國(guó)流行株B亞型gp120,gag基因在重組痘苗病毒中的表達(dá)及其實(shí)驗(yàn)免疫研究[J];中國(guó)輸血雜志;2001年S1期

9 宋艷輝;邢輝;王哲;張遠(yuǎn)志;孫峰;何翔;陳建平;邵一鳴;;河南和新疆地區(qū)HIV-1毒株gp120和gag基因的變異分析[J];中國(guó)艾滋病性病;2007年05期

10 周曉蘭;何翔;洪坤學(xué);王哲;邢愛(ài)華;阮玉華;陳健平;邢輝;邵一鳴;;我國(guó)河南、陜西兩省獻(xiàn)血感染者HIV-1B亞型毒株gag基因變異研究[J];病毒學(xué)報(bào);2009年02期

相關(guān)碩士學(xué)位論文 前3條

1 楊文斌;豬內(nèi)源性反轉(zhuǎn)錄病毒gag基因的克隆及其在大腸桿菌中的表達(dá)[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2003年

2 初向華;扇貝裙邊GAG對(duì)堿性成纖維細(xì)胞生長(zhǎng)因子誘導(dǎo)的血管平滑肌細(xì)胞增殖的影響[D];青島大學(xué);2004年

3 周曉蘭;中國(guó)HIV-1B’毒株gag基因變異特征研究[D];中國(guó)疾病預(yù)防控制中心;2009年

，

本文編號(hào)：1521185