本文關(guān)鍵詞： BVDV 納米抗體 篩選 原核表達(dá) 復(fù)制　出處：《石河子大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV)主要引起牛病毒性腹瀉-粘膜病(BVD-MD),該病尚無完全有效的防制措施,對養(yǎng)牛業(yè)造成巨大的損失。駱駝體內(nèi)發(fā)現(xiàn)存在一種天然缺失輕鏈的重鏈抗體,僅由其可變區(qū)組成的抗體稱為單域抗體,又稱為納米抗體。納米抗體具有高親和力、高穩(wěn)定性、強(qiáng)組織穿透性、高效表達(dá)等優(yōu)點。構(gòu)建抗BVDV納米抗體基因文庫,利用噬菌體展示技術(shù)獲得高表達(dá)、特異性強(qiáng)的納米抗體,為防治BVDV奠定基礎(chǔ)。本文的主要研究內(nèi)容如下:1、BVDV納米抗體基因文庫的庫容量和多樣性鑒定。本實驗采用稀釋計算法測定已構(gòu)建的文庫庫容量,隨機(jī)挑取24個單菌落進(jìn)行菌液PCR對抗體文庫的重組率鑒定,陽性單克隆測序;利用MEGA 4.0構(gòu)建系統(tǒng)進(jìn)化樹、DNAMAN分析氨基酸序列同源性,評價基因文庫的多樣性。結(jié)果表明基因文庫庫容達(dá)到1.22×106、重組率約83.3%、氨基酸序列同源性為63.51%,說明文庫在保存過程中其庫容量沒有受到環(huán)境的影響,多樣性豐富。2、BVDV納米抗體文庫的篩選和克隆鑒定。利用M13K07輔助噬菌體對基因文庫拯救得到噬菌體展示文庫,測定其滴度。以BVDV全病毒作為目標(biāo)抗原,將其結(jié)合于固相上,加入噬菌體展示文庫,經(jīng)三輪“吸附-洗脫-擴(kuò)增”親和篩選后,隨機(jī)挑取單克隆進(jìn)行ELISA檢測。結(jié)果拯救的噬菌體展示文庫滴度約1.2×1013 cfu/mL,三輪篩選使噬菌體文庫中的特異性單克隆得到有效的富集,ELISA檢測得到1株陽性克隆,命名為VHH-A6。3、BVDV納米抗體的原核表達(dá)和功能驗證。根據(jù)VHH-A6基因的測序序列,設(shè)計引物,采用PCR擴(kuò)增技術(shù)克隆VHH-A6基因,構(gòu)建pET-28a(+)-VHH-A6重組原核表達(dá)載體,經(jīng)SDS-PAGE檢測目的蛋白,Ni柱純化得到高純度的目的蛋白,在細(xì)胞水平驗證VHH-A6納米抗體對BVDV復(fù)制的影響。結(jié)果表明:成功獲得VHH-A6基因及其重組原核表達(dá)載體,得到高純度的目的蛋白。利用實時熒光定量PCR(RT-q PCR)測定MDBK細(xì)胞內(nèi)的病毒拷貝數(shù),結(jié)果發(fā)現(xiàn)100μg/mL的VHH-A6納米抗體對BVDV的復(fù)制有一定阻斷效果。本研究通過對BVDV納米抗體文庫進(jìn)行三輪親和篩選,從中篩選獲得與BVDV特異性結(jié)合的納米抗體VHH-A6片段,對其進(jìn)行原核表達(dá)并純化,細(xì)胞水平驗證VHH-A6對BVDV的復(fù)制有一定的阻斷效果,為進(jìn)一步探討納米抗體干擾病毒復(fù)制機(jī)理奠定基礎(chǔ)。
[Abstract]:Bovine viral diarrhea virus (BVDVV) is the main cause of bovine viral diarrhoea-mucosal disease (BVD-MDV), which has not been prevented completely and has caused huge losses to cattle industry. A natural light chain antibody was found in camels. The antibody composed only by its variable region is called single domain antibody, also called nanometer antibody. It has the advantages of high affinity, high stability, strong tissue penetration, high expression and so on. Using phage display technology to obtain high expression, strong specificity of nano-antibody, The main contents of this paper are as follows: 1) Identification of the capacity and diversity of the gene library of BVDV nanoscale antibody. The capacity of the constructed library was measured by dilution calculation method. Twenty-four single colonies were randomly selected to identify the recombination rate of the antibody library by PCR, and the positive monoclonal DNA sequence was sequenced. The phylogenetic tree DNA man was constructed with MEGA 4.0 to analyze the amino acid sequence homology. The results showed that the capacity of the gene library was 1.22 脳 10 ~ 6, the recombination rate was about 83.3and the amino acid sequence homology was 63.51, which indicated that the storage capacity of the library was not affected by the environment. Screening and cloning of multiplex BVDV nano-antibody library. The phage display library was obtained by using M13K07 auxiliary phage to rescue the gene library, and its titer was determined. BVDV whole virus was used as the target antigen to bind it to the solid phase. Add phage display library, after three rounds of "adsorption-eluation-amplification" affinity screening, Results the titer of the rescued phage display library was about 1.2 脳 1013 cfu-mL. three rounds of screening showed that the specific monoclonal antibody in the phage library was effectively enriched by Elisa, and a positive clone was obtained by Elisa. Prokaryotic expression and functional verification of BVDV nano-antibody named VHH-A6.3. according to the sequence of VHH-A6 gene, primer was designed, VHH-A6 gene was cloned by PCR amplification technique, and the recombinant prokaryotic expression vector pET-28a (pET-28a- VHH-A6) was constructed. The high purity target protein was purified by SDS-PAGE, and the effect of VHH-A6 nano-antibody on BVDV replication was verified at the cell level. The results showed that the VHH-A6 gene and its recombinant prokaryotic expression vector were successfully obtained. High purity target protein was obtained. The viral copy number in MDBK cells was determined by real-time fluorescence quantitative PCR(RT-q PCR. The results showed that 100 渭 g / mL of VHH-A6 nanoparticles could block the replication of BVDV. In this study, three rounds of affinity screening of BVDV nano-antibody library were carried out to obtain VHH-A6 fragments specifically bound to BVDV. The prokaryotic expression and purification were carried out to verify the blocking effect of VHH-A6 on the replication of BVDV at cell level, which laid a foundation for further study on the mechanism of interference of virus replication by nano-antibody.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.4

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本文編號：1550361