本文選題：豬流行性腹瀉　切入點(diǎn)：遺傳變異　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)引起感染豬表現(xiàn)為嘔吐、發(fā)熱、脫水、嚴(yán)重腹瀉的一種高度接觸性腸道傳染病,發(fā)病主要集中在冬季,可感染各階段豬,但以哺乳仔豬發(fā)病和病死率最高,可達(dá)100%,給全世界養(yǎng)豬業(yè)造成巨大經(jīng)濟(jì)損失。目前沒有有效的防控措施,有研究表明,接種過疫苗的豬群照樣有暴發(fā)PED的情況,到2010年這一現(xiàn)象更為嚴(yán)重。因此,建立檢測(cè)PEDV抗體水平的診斷方法很有必要。本試驗(yàn)對(duì)PEDV N基因進(jìn)行了克隆和原核表達(dá),用純化重組N蛋白為包被抗原,建立了PEDV血清抗體水平間接ELISA檢測(cè)方法并初步應(yīng)用于臨床,獲得如下研究結(jié)果:1.采集陜西省部分地區(qū)豬場(chǎng)疑似感染PEDV死亡的豬小腸樣品5份,用RT-PCR擴(kuò)增5份樣品的N、S和M基因,分別命名為1-SL、2-BJ、3-YL、4-WN和5-HZ。同源性比較分析,本省流行的5個(gè)毒株與中國(guó)現(xiàn)用疫苗株CV777的S、M和N基因中S基因同源相似性最低,核苷酸相似性為94.9%~99.2%,氨基酸序列相似性為94.9%~99.7%,其次是N基因,核苷酸同源相似性為95.1%~99.9%,氨基酸序列相似性為96.2%~100%,M基因同源相似性最高,氨基酸序列相似性為96.2%~100%;遺傳進(jìn)化樹分析,5個(gè)毒株的S、M和N基因均與疫苗株CV777遺傳親緣關(guān)系相對(duì)較遠(yuǎn);同一省份不同地區(qū)5株流行株的不同基因遺傳親緣遠(yuǎn)近不一樣。2.設(shè)計(jì)N基因的一對(duì)特異性引物,提取病毒RNA,以HiScript?Q RT SuperMix for qPCR說明書反轉(zhuǎn)錄的cDNA為模版,PCR擴(kuò)增和核酸膠回收目的基因,克隆N基因并原核表達(dá),重組質(zhì)粒pET-28a-N轉(zhuǎn)化至大腸埃希菌BL21。重組蛋白分子質(zhì)量約58 kD,SDS-PAGE分析為可溶性蛋白。用優(yōu)質(zhì)鎳柱純化裂解上清液,獲得量大、純度較高的蛋白,Western blot分析與PEDV陽(yáng)性血清有良好的特異反應(yīng)性。用純化N蛋白為包被抗原,ELISA檢測(cè)最優(yōu)條件為:抗原包被量每孔0.5μg/mL,血清以1:200倍稀釋作用1.0h,酶標(biāo)二抗以1:5000稀釋作用1.0 h,TMB顯色時(shí)間25 min。敏感性、特異性、重復(fù)性檢測(cè)均良好,初步檢測(cè)臨床164份血清,效果較好。
[Abstract]:Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection caused by porcine epidemic diarrhea virus (PEDVV), which is characterized by vomiting, fever, dehydration and severe diarrhea in pigs. However, the suckling piglets have the highest morbidity and fatality rate, which can reach 100, causing huge economic losses to the world pig industry. There are no effective prevention and control measures at present. Studies have shown that vaccinated pigs still have PED outbreaks. By 2010, this phenomenon is more serious. Therefore, it is necessary to establish a diagnostic method to detect the level of PEDV antibody. In this study, the PEDV N gene was cloned and expressed in prokaryotic cells, and the purified recombinant N protein was used as the coated antigen. The indirect ELISA detection method of PEDV serum antibody level was established and applied to clinical practice. The following results were obtained: 1. Five small intestine samples of pigs suspected to be infected with PEDV were collected from pig farms in some parts of Shaanxi Province, and 5 samples were amplified by RT-PCR. The homology analysis showed that the homology of S gene was the lowest, the nucleotide similarity was 94.9 ~ 99.2, amino acid sequence similarity was 94.9m ~ 99.2, amino acid sequence similarity was 94.9m ~ 99.7, followed by N gene. The homology of nucleotide sequence and amino acid sequence was 95.1% and 99.9, respectively. The similarity of amino acid sequence and amino acid sequence was the highest, and the similarity of amino acid sequence was 96.20.The genetic phylogenetic tree analysis showed that the Sm and N genes of the five virulent strains were relatively far related to the CV777 of the vaccine strain. The genetic relationship of different genes of 5 epidemic strains in the same province is different. 2. Design a pair of specific primers of N gene to extract virus RNAs and use HiScript? The reverse transcription cDNA of Q RT SuperMix for qPCR was the target gene of template PCR amplification and nucleic acid gel recovery, the N gene was cloned and expressed in prokaryotic cells. The recombinant plasmid pET-28a-N was transformed into Escherichia coli BL21. The recombinant protein was analyzed into soluble protein by SDS-PAGE with a molecular weight of about 58 kD. The supernatant was purified by high quality nickel column. Western blot analysis of protein with high purity and PEDV positive serum showed good specific reactivity. The optimal conditions for detection of purified N protein by Elisa were as follows: antigen encapsulation volume was 0.5 渭 g / mL, serum was diluted by 1: 200 times for 1.0 h, and enzyme labeled second antibody was used as Elisa method. Sensitivity of 1: 5000 diluted to 1.0 h TMB for 25 mins, The specificity and repeatability were all good, and 164 serum samples were detected preliminarily.
【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
【分類號(hào)】：S858.28

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