人工设计骆驼源单域抗体库构建及雌二醇抗体筛选

发布时间：2018-01-01 01:13

本文关键词：人工设计骆驼源单域抗体库构建及雌二醇抗体筛选　出处：《中国人民解放军军事医学科学院》2016年硕士论文　论文类型：学位论文

【摘要】：食品安全是全球关注的热点问题之一,其被排在中国人最担心的五个安全问题的第一位,超过了公共安全、交通安全、健康安全和环境安全。加强食品安全危害因子检测是解决食品安全的重要手段。对于食品安全检测中的免疫检测方法来说,针对特定的靶标快速制备亲和力好且背景清晰的特异性抗体是解决免疫检测方法特异性和一致性主要工作之一。因此,本研究以骆驼源单域抗体为基础,采用基因测序和计算生物学的方法,分别以实体抗体库和虚拟抗体库两种形式建立了骆驼源单域抗体库,并以典型的雌激素类物质雌二醇作为靶标,通过筛选雌二醇特异性的单域抗体初步验证了该抗体筛选平台的可行性,利用间接ELISA和SPR验证了特异性抗体的结合活性、竞争活性以及亲和力,成功构建了基于计算生物学方法的骆驼源单域抗体库,并针对雌二醇抗体的筛选。1.基于第一代测序技术的单域抗体基因库的构建首先从健康成年双峰驼的颈静脉采取外周血液,分离其单核淋巴细胞,用TRIZOL法提取淋巴细胞中的总mRNA。通过反转录试剂盒,以Oligo T作为扩增引物、总mRNA作为模板合成cDNA第一条链。通过设计好的上游引物及下游引物,利用PCR反应从cDNA模板中扩增得到VHH的基因片段。以pMD18-T为扩增质粒,通过TA克隆技术,将VHH基因片段克隆到pMD18-T载体中。通过比较VH和VHH的保守FR2区域的37、44、45、47号位点氨基酸的差异,查找VH的基因片段并删除。最后,将筛选好的VHH基因片段进行同源性分析,并通过生成的进化树对氨基酸序列完全相同的VHH进行删除。初步构建了含2000株天然抗体的抗体库。通过健康成年双峰驼的外周血单核淋巴细胞建立得到天然骆驼源单域抗体的基因库,并通过测序结果的序列比对剔除了VHH基因片段中传统抗体的VH基因片段,利用同源性分析删除重复的VHH核酸序列,保证了该天然抗体库的抗体多样性。抗体库中的每一株VHH都具有完整的核酸序列以及氨基酸序列,并以pMD18-T-VHH重组质粒的形式进行了保存。2.完成了建模选择、参数筛选等关键计算生物学技术平台构建,建立了虚拟抗体库在第一部分实验构建的单域抗体基因库的基础上,通过引入同源建模和分子动力学模拟的计算生物学方法,构建了单域抗体虚拟库。首先,将每一株vhh抗体的氨基酸序列提交到abnum网站上进行抗体编号,通过编号识别了每株抗体的互补决定区(cdr)和框架区(fr)区。编号完成后,将每一株vhh抗体的氨基酸序列提交到swiss-model网站上进行在线的同源建模。同源建模属于比较建模方法的一种,该方法在蛋白质分子结构预测中常用,也比较可靠。针对vhh抗体中cdr区结构的不稳定性,在下一步实验中,对每个vhh模型进行了分子动力学模拟来对每个vhh三个cdr区的loop结构进行了优化,模拟过程选择操作较为简单的gromacs软件进行。为解决分子动力学模拟过程所消耗的大量计算资源,实验中借助了天河一号国家计算中心的超级计算机,以远程操作的方式完成了该实验步骤。通过优化模拟步骤,对每个vhh模型首先进行了能量最小化、“等温-等容”平衡、“等温-等压”平衡等过程,释放了vhh结构内部的势能并平衡了外周环境的温度和压力,最终进行了10ns的模拟时长。模拟结束后,以rmsd分析和gyrate分析为标准对vhh的模型进行评估并选择。本实验中,通过抗体编号完成了不同结构域的识别。在抗体的可变区中,fr区的氨基酸种类和排序较为保守,其结构常常折叠成为抗体可变区的骨架,而三个cdr区则形成不规则的loop结构。对vhh抗体同源建模的结果进行分析发现,在对10个模型进行比较时,发现每个模型之间的三个cdr区的结构在10个模型之间的差异很大。因此在利用分子动力学模拟的方法对vhh抗体的模型进行了优化,在模拟过程中,vhh结构中由于不恰当的几何构象和空间位阻所储存的能量得到了释放,并且平衡了外周环境的温度参数和压力参数,整个体系的参数得到了保证。分子动力学模拟结束后,通过rmsd和gyrate的分析发现,vhh抗体在模拟的过程中,结构变化逐渐趋于稳定,使其蛋白的结构折叠地更为紧凑。通过上述过程,虚拟抗体库中的2000株抗体均获得了可靠的三维结构,并在抗体编号和结构比对结果的基础上,不同结构域有了清楚的划分,这便于后续分子对接实验的进行和分析等过程。通过上述步骤获得了较为可靠的vhh抗体三维结构,并对氨基酸在fr区和cdr区结构域中的位置进行了划分。基于同源建模及分子动力学模拟方法,完成了vhh抗体虚拟抗体库的构建,库中每株vhh都获得了可靠的三维结构并且不同结构域的氨基酸得到了划分。3.建立虚拟筛选策略,优化分子筛选技术,完成了以雌二醇为代表靶标的vhh抗体的虚拟筛选为了初步验证该抗体筛选平台,后续实验中选择了动物性食品中典型的雌激素类污染物雌二醇作为靶标进行研究。对于该抗体平台的虚拟筛选策略,本实验选择了分子对接方法完成了抗体库针对雌二醇小分子的虚拟筛选。在实现分子对接的软件中,autodock是研究领域中使用最多的。本研究中,在对vhh抗体的结构进行准备时,选择三个cdr区所在的空间位置作为雌二醇小分子的对接位范围,将三个loop结构中形成孔穴结构的氨基酸设置为柔性残基,然后用不同类型的原子作为探针,通过运行autogrid程序计算格点能量,最后根据雌二醇小分子的不同结合构象以及能量等参数进行评分,对对接结果进行排序。最后通过结合自由能的数值对雌二醇特异性的vhh抗体进行筛选,并对筛选得到的e2-vhh复合物进行了相互作用模式的分析。对筛选的雌二醇vhh抗体通过分析发现,vhh抗体的三个cdr区形成了合理的抗原结合结构域,为雌二醇小分子的结合提供了有利的空间构象。对e2-vhh复合物进行相互作用的分析时发现,当vhh的cdr区,特别是cdr3区中存在氨基酸残基含有氮元素时,尤其是氨基中的氮元素,其可以通过孤对电子和雌二醇中位于结构两端的羟基氢形成氢键,另外当cdr区含有芳香环,并且和雌二醇中苯环结构的相对空间位置合适时,可以通过形成π-π键来加强雌二醇和vhh抗体的相互作用力。通过上述分析结果发现,通过分子对接技术,可以有效的完成抗体和雌二醇小分子之间的对接分析,实现了vhh虚拟抗体库的虚拟筛选策略。此外通过利用超级计算机,针对抗体库的虚拟筛选,解决了其耗费计算资源的问题,实现了抗体库中2000株vhh抗体的高效虚拟筛选。本筛选方法的建立利用了计算生物学中的分子对接技术,并依托超级计算机实现了vhh虚拟抗体库对雌二醇小分子的快速筛选,与传统的抗体筛选技术相比,在对筛选平台得到的结果进行分析后,得到了雌二醇和vhh抗体之间相互作用模式等重要信息,为vhh抗体的亲和力成熟以及抗体的设计等工作奠定了基础。4.雌二醇vhh抗体的表达、纯化与性质鉴定选择了常用的pet载体系列对vhh蛋白进行了重组表达。首先,设计vhh的扩增引物,在上游引物和下游引物中分别引入bamhi和ecori酶切位点,为了保证酶切的效果,先将扩增好的含有酶切位点的vhh基因片段克隆到pmd18-t质粒中,然后对pmd18-t-vhh质粒以及pet32a质粒进行双酶切处理,通过t4ligase连接酶进行连接,并转化bl21(de3)感受态细胞,通过菌落pcr以及测序结果分析,选择测序结果正确以及开放读码框正确的菌株进行转接培养并保存菌株。在进行大量表达时,首先将菌液进行活化,然后转接到大瓶培养基中,待培养菌液的od600值达到0.5-0.6之间,加入iptg进行诱导表达,37℃,12-15h过夜。通过sds-page检测表达的菌液,vhh具有很高的表达量,通过超声破碎后,VHH以包涵体的形式存在在破碎后离心的沉淀中,利用包涵变复性对VHH进行了高纯度的纯化,并通过BCA蛋白定量试剂盒对纯化后的可溶性VHH进行蛋白定量。在雌二醇VHH抗体的性质鉴定实验中,选择了虚拟筛选结果中结合自由能值最低的一株抗体VHH165进行验证,以间接ELISA和SPR两个实验方法分别对VHH抗体的结合性质、竞争活性以及亲和力常数进行了测定。在雌二醇VHH抗体的间接ELISA实验研究中发现,随着VHH165的浓度增加,和包被在酶联孔中的E2-OVA的结合逐渐增加,并且远高于VHH165和OVA的结合;通过间接ELISA初步验证,VHH165的IC50值为20.03ng/mL,最低检测限LOD:IC10值为4.079ng/mL,通过SPR测定,VHH165的亲和力为1.49mM。本研究应用测序技术及计算生物学方法,建立了骆驼源天然单域抗体库,包括VHH实体库以及VHH虚拟库,并利用分子对接的方法实现了雌二醇VHH抗体的快速虚拟筛选,在小分子与抗体的结合复合物的分析的基础上获得了相互作用模式的信息。此外,还建立了骆驼源单域抗体的高效原核表达体系,并检测了雌二醇特异性抗体的结合性质、竞争活性以及亲和力常数,初步验证了该VHH抗体筛选平台以及虚拟筛选策略。该VHH抗体筛选平台和通过传统方法建立的筛选方法相比有如下两点优势:一方面,该抗体库利用了骆驼源的单域抗体,这类小型化的抗体具有分子量小,热稳定性高、可以耐受复杂应用环境以及易制备等优势,其有助于抗体制备时的基因操作、高水平表达表达以及易于保存等;另一方面,通过借助计算生物学方法,可以实现对抗体库中的每一株抗体的遗传背景、蛋白质二级和三级结构以及特异性抗体和识别靶标相互作用模式的清晰认识。在未来的工作中,通过综合以上信息可以实现对特异性抗体的虚拟设计,并大大提高了抗体制备后期亲和力成熟的速度以及可靠性。在单域抗体库建立后,通过本方法获得的特异性VHH抗体,具有较好的亲和力,以及较为全面的生物背景信息,可以在进一步的抗体制备过程中开展可靠的亲和力成熟及抗体设计等工作。
[Abstract]:Food safety is one of the hot issues of global concern, the first is ranked in the five Chinese most worried about security issues, more than the public safety, traffic safety, health and environmental safety. To strengthen food safety hazard detection is an important means to solve food safety. The immune detection method in detection of food safety speaking, for rapid preparation of good affinity and clear background specific antibody specific target detection method is one of the immunological specificity and consistency of the main work. Therefore, in this study, the camel source single domain antibody based methods using gene sequencing and computational biology, respectively to the real and virtual antibody antibody library two forms of Library established from camel single domain antibody library, and taking the typical estrogen estradiol as the target, through the single domain antibody screening E2 specific preliminary verified The feasibility of the antibody screening platform, verify the binding activity of antibody by indirect ELISA and SPR, the competition activity and affinity, the successful construction of computational biology methods camel source single domain antibody library based on.1., and screening for estradiol antibody construct single domain antibody gene library first generation sequencing technology based on the first healthy adult Bactrian camel's jugular vein to peripheral blood mononuclear cells, its isolation, total mRNA. lymphocytes extracted by reverse transcription kit by TRIZOL method, using Oligo T as primers, total mRNA as a template for the synthesis of first strand cDNA. The upstream primer and downstream primer design, the use of PCR gene was obtained VHH fragments amplified from cDNA template. PMD18-T amplification of plasmid by TA cloning technique, VHH gene fragment was cloned into the pMD18-T vector. Through the comparison of the VH and VHH of the conservative F The difference in area R2 No. 37,44,45,47 amino acid of VH gene fragment, search and delete. Finally, VHH gene fragment screening good homology analysis and phylogenetic tree generated by the amino acid sequence of identical VHH were deleted. Initially constructed antibody library containing 2000 strains of natural antibody gene library establishment. Natural camel source single domain antibody by mononuclear lymphocytes in peripheral blood of healthy adult Bactrian camel, and sequence alignment by sequencing results excluding the VH gene fragment of traditional antibody VHH gene fragment, using homology analysis of nucleic acid sequence repeat deletion of VHH, the antibody of the natural antibody library diversity of each. Strain VHH antibody library has complete nucleic acid sequence and amino acid sequence, and recombinant plasmid pMD18-T-VHH by the form of the preservation of.2. completed the modeling, parameter selection and other key. Is the biological technology platform, established a virtual antibody library based on single domain antibody gene library construction on the first part of the experiment, through computational biology methods into homology modeling and molecular dynamics simulations, constructed the single domain antibody virtual library. First of all, will be submitted to the amino acid sequence of each strain of VHH antibody to abnum website antibody number, identification number per plant through the complementary determining region (CDR) antibody and framework region (FR). Number is completed, will be submitted to the amino acid sequence of each strain of VHH antibody to homologous modeling online on the SWISS-MODEL website. A comparison of the homology modeling belongs to the modeling method, the method in protein molecules structure prediction commonly used, reliable. According to the structure of CDR VHH antibody in the instability in the next step, for each VHH model were investigated by molecular dynamics simulation for each VHH three CD The structure of loop R region were optimized, the simulation process selection operation is relatively simple GROMACS software. In order to solve the molecular dynamics simulation of a large amount of computing resources consumed by the process, experiment with supercomputer Tianhe National Computing Center, the remote operation mode to complete the experimental steps. By optimizing the simulation steps for each VHH model firstly, energy minimization, "isothermal - Volume" balance, "isothermal isopiestic equilibrium" process, the release of the VHH structure of the internal potential and temperature and pressure balance of the peripheral environment, finally carried on the 10ns simulation. Simulation after using RMSD analysis and gyrate analysis for the standard of VHH model for evaluation and selection. In this experiment, the number of antibodies to complete the identification of different domains. In the antibody variable region, FR region of amino acids and sorting is more conservative, the The structure often folded into antibody variable region of the skeleton, and the three CDR region is formed loop irregular structure. Antibody to VHH homology modeling results showed that in comparison of 10 models, found three CDR regions of each model between the difference between the 10 models. So great in the method of using molecular dynamics simulation of VHH antibody model were optimized in the simulation process, the structure of VHH due to inappropriate geometry conformation and steric hindrance of the stored energy has been released, and balance the temperature parameters and pressure parameters of the peripheral environment, parameters of the whole system is guaranteed. Molecular dynamics simulation after the analysis of RMSD and gyrate, VHH antibodies in the simulation process, the structure changes gradually stabilized, the structure of the protein folding is more compact. Through the above process, virtual Antibody library of 2000 strains of antibodies were obtained reliable three-dimensional structures, and on the basis of antibody number and structure comparison results, different domains have a clear division, and the analysis process for subsequent molecular docking experiments. VHH antibody can rely on the three-dimensional structure is obtained by the above steps, and the position of amino acids in FR and CDR domains were divided. Homology modeling and molecular dynamics simulation method based on the constructed virtual VHH antibody antibody library, the library VHH per plant was reliable and the three-dimensional structure of amino acid of different domains are divided.3. to establish a virtual screening strategy, optimization molecular screening technology, completed the virtual screening VHH antibody to estradiol as the representative of the target in order to validate the antibody screening platform, follow-up experiment chose the animal food typical estrogen Research on pollutant estradiol as a target. The antibody screening strategy for the virtual platform, virtual screening was chosen in this experiment the molecular docking method to complete the antibody library for estradiol small molecules. In the realization of molecular docking software, autodock is the most commonly used in the research field. In this study, were prepared in the structure of VHH antibody, space location three CDR area as the docking of small molecules of estradiol range, will form three loop structure of amino acids in the cavity structure is set to flexible residues, and then use the original sub different types as probes, by running the autogrid program to calculate the lattice energy, finally according to the score estradiol of small molecules with different conformations and energy parameters, to sort the docking results. Finally, by combining numerical sieve VHH free energy on estradiol antibody specificity Selection of the e2-vhh composites were obtained and analyzed the interaction mode. The estradiol VHH antibody screening through the analysis found that three CDR VHH antibody forming a reasonable antigen binding domain, provides favorable space conformation for binding of estradiol. Analysis of small molecules found each other the role of the e2-vhh complex, when the VHH CDR area, especially nitrogen contained amino acid residues exist in CDR3 region, especially in the amino nitrogen, which can be realized by the lone pair hydroxyl hydrogen structure located at both ends of the electron and estradiol in the formation of hydrogen bond, in addition to the CDR region containing aromatic ring. And the relative location of estradiol in the benzene ring structure when appropriate, can strengthen the interaction of estradiol and VHH antibody by forming pi pi bond. Based on the above analysis results, through molecular docking technology, can effectively complete the anti Analysis of connection between body and estradiol of small molecules, to achieve a virtual screening strategy for virtual VHH antibody library. In addition by using a super computer for virtual screening of antibody library, solve the cost of computing resources, to achieve efficient virtual screening of antibody library of 2000 strains of VHH antibody. The establishment of the screening method of use the calculation of molecular docking technology in biology, and achieve a rapid screening of small molecule VHH estradiol antibody library based on virtual super computer, compared with the traditional antibody screening technology in screening of platform results in the analysis, obtained between estradiol and VHH antibody interaction mode and other important information, which the expression of.4. of estradiol VHH antibody VHH antibody affinity maturation and antibody design work, purification and characterization of the commonly used PET series of carrier protein VHH The recombinant expression. First, design primers VHH, upstream primer and downstream primer were introduced by BamHI and EcoRI restriction sites, in order to ensure the digestion effect, first amplified containing restriction sites of the VHH gene fragment was cloned into pmd18-t plasmid, then pmd18-t-vhh plasmid and pet32a plasmid for double enzyme treatment, are linked by t4ligase ligase (DE3), and transformed into BL21 competent cells by colony PCR and sequencing analysis, selection and sequencing results correct open reading frame right transfer training and save the strain strain. After a great deal of expression, the bacteria were activated, and then transferred to the a large bottle of medium to be cultured bacteria liquid OD600 value reached 0.5-0.6, adding IPTG induced expression, 37 C, 12-15h overnight. Bacteria detected by SDS-PAGE expression, VHH expression is very high, By sonication, VHH in the form of inclusion body in centrifugal broken after precipitation, denaturation and renaturation of inclusion by VHH were purified with high purity, and through the BCA protein assay kit protein. After purification of the soluble VHH in the experimental characterization of estradiol VHH antibody, binding free the use of virtual screening results in a minimum value VHH165 antibody verified by indirect ELISA and SPR two experimental methods were used to VHH antibody binding properties, competitive activity and affinity constants were determined. It was found that in the experimental study on indirect ELISA estradiol VHH antibody, with the increase of VHH165 concentration, and package when combined enzyme linked hole in the E2-OVA gradually increased, and much higher than the combination of VHH165 and OVA by indirect ELISA; preliminary validation, VHH165 IC50 value is 20.03ng/mL, the minimum detection limit was 4.079ng/mL LOD:IC10, Through the determination of SPR, VHH165 affinity for the 1.49mM. based on the sequencing technology and computational biology methods, established the camel source natural single domain antibody library, including VHH entity library and VHH virtual library, and by using molecular docking to achieve rapid virtual screening of estradiol VHH antibody, obtained the interaction modes of information in a small molecular and antibody based on the analysis of the complexes. In addition, also established a prokaryotic expression system of camel source single domain antibody, and to detect the binding properties of estradiol antibody specificity, competitive activity and affinity constants, preliminary verification of the VHH antibody screening platform and the virtual screening strategy. VHH antibody screening platform compared with the screening method established by traditional methods have the following advantages: on the one hand, the use of single domain antibody antibody library, Luo Tuoyuan, this kind of miniaturized with antibody Small molecular weight, high thermal stability, can withstand complex application environment and easy preparation, the genetic manipulation helps antibody preparation of high level expression and easy to store; on the other hand, by means of computational biology methods, can be achieved on the antibody library of the genetic background of each strain antibody the clear understanding of protein two level and three level structure and specific antibody and identification of target interaction mode. In the future work, the above information can realize the virtual design of specific antibody, and greatly improve the preparation of antibody affinity maturation rate and the late

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;R155.5

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