本文選題：新城疫病毒 + 納米抗體��； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：新城疫(Newcastle disease,ND)是由新城疫病毒(NDV)引起禽類的一種嚴(yán)重的高度接觸性傳染病。雖然對(duì)新城疫采取疫苗接種的預(yù)防措施,但是疫苗并不能完全保護(hù)免疫雞群免受感染。因此,研發(fā)一種有效防控新城疫的新方法勢在必行。而融合蛋白(F蛋白)與病毒的毒力和致病性密切相關(guān),作為主要的保護(hù)性抗原,靶向F蛋白的抗體可以有效的阻止病毒與細(xì)胞的融合進(jìn)而保護(hù)雞群免受NDV感染,在NDV的研究中具有非常重要的意義。通過基因工程方法克隆獲得的重鏈抗體可變區(qū)(variable domain of heavy chain of heavy-chain antibody,VHH)是目前已知的具有完整功能的最小抗體分子片段,由于納米抗體較傳統(tǒng)抗體具有一些獨(dú)特的性質(zhì):分子量小、容易制造和表達(dá)、高水溶性和穩(wěn)定性、能識(shí)別獨(dú)特的構(gòu)造表位、具有較高的親和力,使得其在病毒病的診斷和治療等領(lǐng)域的應(yīng)用得到廣泛關(guān)注。本實(shí)驗(yàn)應(yīng)用酵母雙雜交系統(tǒng)從天然納米抗體庫中規(guī)�；Y選出針對(duì)新城疫病毒F蛋白的納米抗體,并對(duì)功能進(jìn)行初步的鑒定,從而為利用納米抗體防控新城疫提供新的思路。主要的研究內(nèi)容如下:1.雙峰駝天然納米抗體酵母雙雜交文庫的構(gòu)建。采集雙峰駝外周血、骨髓、脾臟和淋巴結(jié),并從中分離外周血淋巴細(xì)胞。經(jīng)抽提總RNA,反轉(zhuǎn)錄為cDNA,用2對(duì)引物經(jīng)過2輪PCR,用RT-PCR方法特異性擴(kuò)增得到400 bp左右的雙峰駝重鏈抗體可變區(qū)(VHH)片段。通過酵母雙雜交技術(shù),根據(jù)Clontech公司的文庫構(gòu)建說明書構(gòu)建雙峰駝天然納米抗體文庫。構(gòu)建的雙峰駝天然納米抗體酵母雙雜交文庫庫容和滴度分別達(dá)到2.07×107和7.6×108 cfu/mL,文庫的重組率達(dá)到91%以上,符合酵母雙雜交文庫構(gòu)建要求,為進(jìn)一步獲得特定抗原的VHH奠定了基礎(chǔ)。2.新城疫病毒F蛋白靶向納米抗體的篩選。將合成F蛋白的線性中和表位區(qū),克隆至酵母雙雜交系統(tǒng)誘餌載體pGBKT7中,經(jīng)酶切鑒定和測序驗(yàn)證正確后,獲得重組誘餌質(zhì)粒pGBKT7-F-neu,檢測其在酵母細(xì)胞中的毒性和自激活活性。利用酵母雙雜交的方法對(duì)已構(gòu)建的雙峰駝天然納米抗體文庫進(jìn)行篩選,并對(duì)獲得的陽性克隆進(jìn)行鑒定。3.納米抗體的表達(dá)與功能鑒定。將經(jīng)驗(yàn)證正確的序列插入pHISE原核表達(dá)載體中,構(gòu)建納米抗體表達(dá)質(zhì)粒pHISE-VHH。將酶切和測序鑒定均正確的表達(dá)載體轉(zhuǎn)化大腸桿菌Rosetta表達(dá)菌進(jìn)行納米抗體的誘導(dǎo)表達(dá)和純化。通過用NDV La Sota毒包被96孔反應(yīng)板,與VHH進(jìn)行間接ELISA反應(yīng),經(jīng)鑒定2株VHHs均與NDV La Sota毒株反應(yīng),但是反應(yīng)強(qiáng)弱有差異。Western blot試驗(yàn)結(jié)果顯示,2株VHHs能識(shí)別天然NDV表達(dá)的F蛋白。通過病毒微量中和試驗(yàn)證明有2株VHHs均有病毒中和活性,可以有效阻斷NDV(F48E9株)對(duì)雞胚成纖維細(xì)胞的感染。
[Abstract]:Newcastle disease (NDV) is a severe highly contagious disease in poultry caused by Newcastle disease virus (NDV). Although vaccinated against Newcastle disease, vaccines do not fully protect chickens from infection. Therefore, it is imperative to develop a new method to prevent and control Newcastle disease. The fusion protein F) is closely related to the virulence and pathogenicity of the virus. As the main protective antigen, the antibody targeting F protein can effectively prevent the fusion of virus and cells and protect chickens from NDV infection. It is of great significance in the research of NDV. The variable domain of heavy chain of heavy-chain antibody VHH fragment obtained by genetic engineering method is the smallest known fragment with complete function. Because of the unique properties of nano-antibody compared with traditional antibody, the molecular weight of the antibody is small. It is easy to manufacture and express, highly water-soluble and stable, can recognize unique structural epitopes, and has high affinity, so it has been widely used in the diagnosis and treatment of viral diseases. In this experiment, yeast two-hybrid system was used to screen the nano-antibody against Newcastle disease virus F protein from natural nano-antibody library on a large scale, and the function was preliminarily identified, thus providing a new idea for the prevention and control of Newcastle disease by using nano-antibody. The main contents of the study are as follows: 1: 1. Construction of yeast Two-hybrid Library of Bactrian Camel Natural nanometer Antibody. Peripheral blood, bone marrow, spleen and lymph nodes were collected from Bactrian Camel and peripheral blood lymphocytes were isolated. The total RNAs were extracted and reversely transcribed into cDNA.Two pairs of primers were used to amplify the VHHH fragment of the variable region of the heavy chain antibody of Bactrian Camel by RT-PCR method. The natural antibody library of Bactrian Camel was constructed by yeast two-hybrid technique according to the construction instructions of Clontech Company. The library volume and titer of yeast two-hybrid library of natural nano-antibody of Bactrian camel were 2.07 脳 10 ~ 7 and 7.6 脳 10 ~ 8 cfu-mL, respectively. The recombinant rate of the library was over 91%, which met the requirements of yeast two-hybrid library construction and laid a foundation for further obtaining VHH of specific antigen. Screening of Newcastle Disease virus F protein targeting nanometer Antibodies. The linear neutralizing epitopes of the synthesized F protein were cloned into yeast two-hybrid system bait vector pGBKT7. The recombinant bait plasmid pGBKT7-F-neuwas obtained by restriction enzyme digestion and sequencing. The toxicity and self-activation activity of the recombinant bait plasmid pGBKT7-F-neu in yeast cells were determined. The natural nano-antibody library of Bactrian camel was screened by yeast two-hybrid method, and the positive clone was identified. 3. Expression and functional identification of nano-antibody. The correct sequence was inserted into the prokaryotic expression vector of pHISE and the expression plasmid pHISE-VHHH was constructed. The expression vector was transformed into Escherichia coli Rosetta expression strain by enzyme digestion and sequencing to induce the expression and purification of nano-antibody. Indirect ELISA reaction was carried out with NDV La Sota virus coated with 96 well reaction plate. Two VHHs strains were identified to react with NDV La Sota virus strain, but the reaction intensity was different. Western blot test showed that VHHs could recognize F protein expressed by natural NDV. Virus neutralization test showed that two strains of VHHs had viral neutralization activity, which could effectively block the infection of chicken embryo fibroblasts by NDV(F48E9 strain.
【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】：2015
【分類號(hào)】：S855.3

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本文編號(hào)：1917692