新城疫病毒融合蛋白纳米抗体的筛选与鉴定

发布时间：2018-05-21 04:14

本文选题：新城疫病毒 + 纳米抗体　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：新城疫(Newcastle disease,ND)是由新城疫病毒(NDV)引起禽类的一种严重的高度接触性传染病。虽然对新城疫采取疫苗接种的预防措施,但是疫苗并不能完全保护免疫鸡群免受感染。因此,研发一种有效防控新城疫的新方法势在必行。而融合蛋白(F蛋白)与病毒的毒力和致病性密切相关,作为主要的保护性抗原,靶向F蛋白的抗体可以有效的阻止病毒与细胞的融合进而保护鸡群免受NDV感染,在NDV的研究中具有非常重要的意义。通过基因工程方法克隆获得的重链抗体可变区(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体分子片段,由于纳米抗体较传统抗体具有一些独特的性质:分子量小、容易制造和表达、高水溶性和稳定性、能识别独特的构造表位、具有较高的亲和力,使得其在病毒病的诊断和治疗等领域的应用得到广泛关注。本实验应用酵母双杂交系统从天然纳米抗体库中规模化筛选出针对新城疫病毒F蛋白的纳米抗体,并对功能进行初步的鉴定,从而为利用纳米抗体防控新城疫提供新的思路。主要的研究内容如下:1.双峰驼天然纳米抗体酵母双杂交文库的构建。采集双峰驼外周血、骨髓、脾脏和淋巴结,并从中分离外周血淋巴细胞。经抽提总RNA,反转录为cDNA,用2对引物经过2轮PCR,用RT-PCR方法特异性扩增得到400 bp左右的双峰驼重链抗体可变区(VHH)片段。通过酵母双杂交技术,根据Clontech公司的文库构建说明书构建双峰驼天然纳米抗体文库。构建的双峰驼天然纳米抗体酵母双杂交文库库容和滴度分别达到2.07×107和7.6×108 cfu/mL,文库的重组率达到91%以上,符合酵母双杂交文库构建要求,为进一步获得特定抗原的VHH奠定了基础。2.新城疫病毒F蛋白靶向纳米抗体的筛选。将合成F蛋白的线性中和表位区,克隆至酵母双杂交系统诱饵载体pGBKT7中,经酶切鉴定和测序验证正确后,获得重组诱饵质粒pGBKT7-F-neu,检测其在酵母细胞中的毒性和自激活活性。利用酵母双杂交的方法对已构建的双峰驼天然纳米抗体文库进行筛选,并对获得的阳性克隆进行鉴定。3.纳米抗体的表达与功能鉴定。将经验证正确的序列插入pHISE原核表达载体中,构建纳米抗体表达质粒pHISE-VHH。将酶切和测序鉴定均正确的表达载体转化大肠杆菌Rosetta表达菌进行纳米抗体的诱导表达和纯化。通过用NDV La Sota毒包被96孔反应板,与VHH进行间接ELISA反应,经鉴定2株VHHs均与NDV La Sota毒株反应,但是反应强弱有差异。Western blot试验结果显示,2株VHHs能识别天然NDV表达的F蛋白。通过病毒微量中和试验证明有2株VHHs均有病毒中和活性,可以有效阻断NDV(F48E9株)对鸡胚成纤维细胞的感染。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

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