本文選題：豬流行性腹瀉病毒 + 噬菌體抗體庫(kù)　； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是豬常見(jiàn)多發(fā)病,給養(yǎng)豬業(yè)造成非常嚴(yán)重的經(jīng)濟(jì)損失。目前,疫苗免疫是防控PED的主要措施,但是疫苗免疫存在免疫空白期,高密度養(yǎng)殖帶來(lái)環(huán)境的深度惡化、疫苗免疫失敗、抗生素濫用,以及某些豬場(chǎng)存在藍(lán)耳病和圓環(huán)病毒病等對(duì)疫苗免疫的抑制,傳統(tǒng)的PED活病毒(弱毒)疫苗自身存在著傳播疾病的風(fēng)險(xiǎn)等因素,對(duì)該病的防控帶來(lái)不利的影響。噬菌體展示技術(shù)是將抗體分了展示于噬菌體表面的技術(shù),能夠針對(duì)特定抗原進(jìn)行篩選并得到特異性抗體。豬流行性腹瀉病毒(porcine epidemic diarrhea virus,PEDV)的主要抗原表位均分布在病毒的纖突蛋白(S蛋白,Spike protein)上。利用噬菌體展示技術(shù)構(gòu)建PEDV S蛋白噬菌體抗體庫(kù),并從抗體庫(kù)中篩選和制備PEDV S蛋白特異性單域抗體,相應(yīng)的單域抗體可用于PED診斷、治療和預(yù)防。本研究中,我們利用PEDV免疫雙峰駝,分離外周血淋巴細(xì)胞后提取總RNA,通過(guò)RT-PCR合成cDNA,再用單域抗體特異性引物進(jìn)行三次擴(kuò)增得到VHH基因。將VHH基因片段與pCANTAB5E載體連接,電轉(zhuǎn)化E.coli TG1,成功構(gòu)建了庫(kù)容為5.49×106的初級(jí)單鏈抗體庫(kù)。并將PEDV關(guān)鍵抗原蛋白S克隆到原核表達(dá)載體pET-28a中,重組質(zhì)粒再轉(zhuǎn)化到感受態(tài)細(xì)胞Transetta-DE3,進(jìn)行可溶性蛋白表達(dá)和純化。并以S蛋白作為抗原對(duì)抗體庫(kù)進(jìn)行3輪富集和篩選,Phage-ELISA篩選和獲得了 S蛋白特異性單域抗體。
[Abstract]:Porcine epidemic diarrhea (PED) is a common disease in pigs, which causes serious economic loss to pig industry. At present, vaccine immunization is the main measure to prevent and control PED, but vaccine immunization exists in blank period, high density culture brings about deep deterioration of environment, vaccine immunization fails, antibiotics abuse. Some factors, such as blue ear disease, circovirus disease and so on, existed in some pig farms, and the traditional PED live virus (attenuated virus) vaccine itself had the risk of spreading disease, which had a negative effect on the prevention and control of the disease. Phage display is a technique that separates antibodies on phage surface and can screen specific antigens and obtain specific antibodies. The main antigenic epitopes of porcine epidemic diarrhea virus (PEV) were distributed on the spike protein of the virus. The phage antibody library of PEDV S protein was constructed by phage display technique, and the specific single domain antibody of PEDV S protein was screened and prepared from the antibody library. The corresponding single domain antibody can be used in the diagnosis, treatment and prevention of PED. In this study, we immunized Bactrian Camels with PEDV, isolated peripheral blood lymphocytes, extracted total RNAs, synthesized cDNAs through RT-PCR, and amplified VHH gene with single domain antibody specific primers for three times. The VHH gene fragment was ligated with pCANTAB5E vector and electrotransformed into E.coli TG1. A primary single-chain antibody library with a storage capacity of 5.49 脳 10 ~ 6 was successfully constructed. The key antigen protein S of PEDV was cloned into the prokaryotic expression vector pET-28a, and the recombinant plasmid was transformed into Transetta-DE3 cells for expression and purification of soluble protein. Using S protein as antigen, the antibody library was enriched and screened by Phage-ELISA for three rounds, and S-protein specific single-domain antibody was obtained.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S858.28

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