BVDV纳米抗体的筛

发布时间：2018-03-01 04:23

本文关键词： BVDV 纳米抗体筛选原核表达复制　出处：《石河子大学》2015年硕士论文　论文类型：学位论文

【摘要】：牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)主要引起牛病毒性腹泻-粘膜病(BVD-MD),该病尚无完全有效的防制措施,对养牛业造成巨大的损失。骆驼体内发现存在一种天然缺失轻链的重链抗体,仅由其可变区组成的抗体称为单域抗体,又称为纳米抗体。纳米抗体具有高亲和力、高稳定性、强组织穿透性、高效表达等优点。构建抗BVDV纳米抗体基因文库,利用噬菌体展示技术获得高表达、特异性强的纳米抗体,为防治BVDV奠定基础。本文的主要研究内容如下:1、BVDV纳米抗体基因文库的库容量和多样性鉴定。本实验采用稀释计算法测定已构建的文库库容量,随机挑取24个单菌落进行菌液PCR对抗体文库的重组率鉴定,阳性单克隆测序;利用MEGA 4.0构建系统进化树、DNAMAN分析氨基酸序列同源性,评价基因文库的多样性。结果表明基因文库库容达到1.22×106、重组率约83.3%、氨基酸序列同源性为63.51%,说明文库在保存过程中其库容量没有受到环境的影响,多样性丰富。2、BVDV纳米抗体文库的筛选和克隆鉴定。利用M13K07辅助噬菌体对基因文库拯救得到噬菌体展示文库,测定其滴度。以BVDV全病毒作为目标抗原,将其结合于固相上,加入噬菌体展示文库,经三轮“吸附-洗脱-扩增”亲和筛选后,随机挑取单克隆进行ELISA检测。结果拯救的噬菌体展示文库滴度约1.2×1013 cfu/mL,三轮筛选使噬菌体文库中的特异性单克隆得到有效的富集,ELISA检测得到1株阳性克隆,命名为VHH-A6。3、BVDV纳米抗体的原核表达和功能验证。根据VHH-A6基因的测序序列,设计引物,采用PCR扩增技术克隆VHH-A6基因,构建pET-28a(+)-VHH-A6重组原核表达载体,经SDS-PAGE检测目的蛋白,Ni柱纯化得到高纯度的目的蛋白,在细胞水平验证VHH-A6纳米抗体对BVDV复制的影响。结果表明:成功获得VHH-A6基因及其重组原核表达载体,得到高纯度的目的蛋白。利用实时荧光定量PCR(RT-q PCR)测定MDBK细胞内的病毒拷贝数,结果发现100μg/mL的VHH-A6纳米抗体对BVDV的复制有一定阻断效果。本研究通过对BVDV纳米抗体文库进行三轮亲和筛选,从中筛选获得与BVDV特异性结合的纳米抗体VHH-A6片段,对其进行原核表达并纯化,细胞水平验证VHH-A6对BVDV的复制有一定的阻断效果,为进一步探讨纳米抗体干扰病毒复制机理奠定基础。
[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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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