本文關(guān)鍵詞：人工設(shè)計(jì)駱駝源單域抗體庫(kù)構(gòu)建及雌二醇抗體篩選　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 單域抗體 抗體庫(kù)技術(shù) 計(jì)算生物學(xué) 分子模擬 雌二醇

【摘要】：食品安全是全球關(guān)注的熱點(diǎn)問(wèn)題之一,其被排在中國(guó)人最擔(dān)心的五個(gè)安全問(wèn)題的第一位,超過(guò)了公共安全、交通安全、健康安全和環(huán)境安全。加強(qiáng)食品安全危害因子檢測(cè)是解決食品安全的重要手段。對(duì)于食品安全檢測(cè)中的免疫檢測(cè)方法來(lái)說(shuō),針對(duì)特定的靶標(biāo)快速制備親和力好且背景清晰的特異性抗體是解決免疫檢測(cè)方法特異性和一致性主要工作之一。因此,本研究以駱駝源單域抗體為基礎(chǔ),采用基因測(cè)序和計(jì)算生物學(xué)的方法,分別以實(shí)體抗體庫(kù)和虛擬抗體庫(kù)兩種形式建立了駱駝源單域抗體庫(kù),并以典型的雌激素類物質(zhì)雌二醇作為靶標(biāo),通過(guò)篩選雌二醇特異性的單域抗體初步驗(yàn)證了該抗體篩選平臺(tái)的可行性,利用間接ELISA和SPR驗(yàn)證了特異性抗體的結(jié)合活性、競(jìng)爭(zhēng)活性以及親和力,成功構(gòu)建了基于計(jì)算生物學(xué)方法的駱駝源單域抗體庫(kù),并針對(duì)雌二醇抗體的篩選。1.基于第一代測(cè)序技術(shù)的單域抗體基因庫(kù)的構(gòu)建首先從健康成年雙峰駝的頸靜脈采取外周血液,分離其單核淋巴細(xì)胞,用TRIZOL法提取淋巴細(xì)胞中的總mRNA。通過(guò)反轉(zhuǎn)錄試劑盒,以O(shè)ligo T作為擴(kuò)增引物、總mRNA作為模板合成cDNA第一條鏈。通過(guò)設(shè)計(jì)好的上游引物及下游引物,利用PCR反應(yīng)從cDNA模板中擴(kuò)增得到VHH的基因片段。以pMD18-T為擴(kuò)增質(zhì)粒,通過(guò)TA克隆技術(shù),將VHH基因片段克隆到pMD18-T載體中。通過(guò)比較VH和VHH的保守FR2區(qū)域的37、44、45、47號(hào)位點(diǎn)氨基酸的差異,查找VH的基因片段并刪除。最后,將篩選好的VHH基因片段進(jìn)行同源性分析,并通過(guò)生成的進(jìn)化樹對(duì)氨基酸序列完全相同的VHH進(jìn)行刪除。初步構(gòu)建了含2000株天然抗體的抗體庫(kù)。通過(guò)健康成年雙峰駝的外周血單核淋巴細(xì)胞建立得到天然駱駝源單域抗體的基因庫(kù),并通過(guò)測(cè)序結(jié)果的序列比對(duì)剔除了VHH基因片段中傳統(tǒng)抗體的VH基因片段,利用同源性分析刪除重復(fù)的VHH核酸序列,保證了該天然抗體庫(kù)的抗體多樣性�？贵w庫(kù)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重組質(zhì)粒的形式進(jìn)行了保存。2.完成了建模選擇、參數(shù)篩選等關(guān)鍵計(jì)算生物學(xué)技術(shù)平臺(tái)構(gòu)建,建立了虛擬抗體庫(kù)在第一部分實(shí)驗(yàn)構(gòu)建的單域抗體基因庫(kù)的基礎(chǔ)上,通過(guò)引入同源建模和分子動(dòng)力學(xué)模擬的計(jì)算生物學(xué)方法,構(gòu)建了單域抗體虛擬庫(kù)。首先,將每一株vhh抗體的氨基酸序列提交到abnum網(wǎng)站上進(jìn)行抗體編號(hào),通過(guò)編號(hào)識(shí)別了每株抗體的互補(bǔ)決定區(qū)(cdr)和框架區(qū)(fr)區(qū)。編號(hào)完成后,將每一株vhh抗體的氨基酸序列提交到swiss-model網(wǎng)站上進(jìn)行在線的同源建模。同源建模屬于比較建模方法的一種,該方法在蛋白質(zhì)分子結(jié)構(gòu)預(yù)測(cè)中常用,也比較可靠。針對(duì)vhh抗體中cdr區(qū)結(jié)構(gòu)的不穩(wěn)定性,在下一步實(shí)驗(yàn)中,對(duì)每個(gè)vhh模型進(jìn)行了分子動(dòng)力學(xué)模擬來(lái)對(duì)每個(gè)vhh三個(gè)cdr區(qū)的loop結(jié)構(gòu)進(jìn)行了優(yōu)化,模擬過(guò)程選擇操作較為簡(jiǎn)單的gromacs軟件進(jìn)行。為解決分子動(dòng)力學(xué)模擬過(guò)程所消耗的大量計(jì)算資源,實(shí)驗(yàn)中借助了天河一號(hào)國(guó)家計(jì)算中心的超級(jí)計(jì)算機(jī),以遠(yuǎn)程操作的方式完成了該實(shí)驗(yàn)步驟。通過(guò)優(yōu)化模擬步驟,對(duì)每個(gè)vhh模型首先進(jìn)行了能量最小化、“等溫-等容”平衡、“等溫-等壓”平衡等過(guò)程,釋放了vhh結(jié)構(gòu)內(nèi)部的勢(shì)能并平衡了外周環(huán)境的溫度和壓力,最終進(jìn)行了10ns的模擬時(shí)長(zhǎng)。模擬結(jié)束后,以rmsd分析和gyrate分析為標(biāo)準(zhǔn)對(duì)vhh的模型進(jìn)行評(píng)估并選擇。本實(shí)驗(yàn)中,通過(guò)抗體編號(hào)完成了不同結(jié)構(gòu)域的識(shí)別。在抗體的可變區(qū)中,fr區(qū)的氨基酸種類和排序較為保守,其結(jié)構(gòu)常常折疊成為抗體可變區(qū)的骨架,而三個(gè)cdr區(qū)則形成不規(guī)則的loop結(jié)構(gòu)。對(duì)vhh抗體同源建模的結(jié)果進(jìn)行分析發(fā)現(xiàn),在對(duì)10個(gè)模型進(jìn)行比較時(shí),發(fā)現(xiàn)每個(gè)模型之間的三個(gè)cdr區(qū)的結(jié)構(gòu)在10個(gè)模型之間的差異很大。因此在利用分子動(dòng)力學(xué)模擬的方法對(duì)vhh抗體的模型進(jìn)行了優(yōu)化,在模擬過(guò)程中,vhh結(jié)構(gòu)中由于不恰當(dāng)?shù)膸缀螛?gòu)象和空間位阻所儲(chǔ)存的能量得到了釋放,并且平衡了外周環(huán)境的溫度參數(shù)和壓力參數(shù),整個(gè)體系的參數(shù)得到了保證。分子動(dòng)力學(xué)模擬結(jié)束后,通過(guò)rmsd和gyrate的分析發(fā)現(xiàn),vhh抗體在模擬的過(guò)程中,結(jié)構(gòu)變化逐漸趨于穩(wěn)定,使其蛋白的結(jié)構(gòu)折疊地更為緊湊。通過(guò)上述過(guò)程,虛擬抗體庫(kù)中的2000株抗體均獲得了可靠的三維結(jié)構(gòu),并在抗體編號(hào)和結(jié)構(gòu)比對(duì)結(jié)果的基礎(chǔ)上,不同結(jié)構(gòu)域有了清楚的劃分,這便于后續(xù)分子對(duì)接實(shí)驗(yàn)的進(jìn)行和分析等過(guò)程。通過(guò)上述步驟獲得了較為可靠的vhh抗體三維結(jié)構(gòu),并對(duì)氨基酸在fr區(qū)和cdr區(qū)結(jié)構(gòu)域中的位置進(jìn)行了劃分�；谕唇＜胺肿觿�(dòng)力學(xué)模擬方法,完成了vhh抗體虛擬抗體庫(kù)的構(gòu)建,庫(kù)中每株vhh都獲得了可靠的三維結(jié)構(gòu)并且不同結(jié)構(gòu)域的氨基酸得到了劃分。3.建立虛擬篩選策略,優(yōu)化分子篩選技術(shù),完成了以雌二醇為代表靶標(biāo)的vhh抗體的虛擬篩選為了初步驗(yàn)證該抗體篩選平臺(tái),后續(xù)實(shí)驗(yàn)中選擇了動(dòng)物性食品中典型的雌激素類污染物雌二醇作為靶標(biāo)進(jìn)行研究。對(duì)于該抗體平臺(tái)的虛擬篩選策略,本實(shí)驗(yàn)選擇了分子對(duì)接方法完成了抗體庫(kù)針對(duì)雌二醇小分子的虛擬篩選。在實(shí)現(xiàn)分子對(duì)接的軟件中,autodock是研究領(lǐng)域中使用最多的。本研究中,在對(duì)vhh抗體的結(jié)構(gòu)進(jìn)行準(zhǔn)備時(shí),選擇三個(gè)cdr區(qū)所在的空間位置作為雌二醇小分子的對(duì)接位范圍,將三個(gè)loop結(jié)構(gòu)中形成孔穴結(jié)構(gòu)的氨基酸設(shè)置為柔性殘基,然后用不同類型的原子作為探針,通過(guò)運(yùn)行autogrid程序計(jì)算格點(diǎn)能量,最后根據(jù)雌二醇小分子的不同結(jié)合構(gòu)象以及能量等參數(shù)進(jìn)行評(píng)分,對(duì)對(duì)接結(jié)果進(jìn)行排序。最后通過(guò)結(jié)合自由能的數(shù)值對(duì)雌二醇特異性的vhh抗體進(jìn)行篩選,并對(duì)篩選得到的e2-vhh復(fù)合物進(jìn)行了相互作用模式的分析。對(duì)篩選的雌二醇vhh抗體通過(guò)分析發(fā)現(xiàn),vhh抗體的三個(gè)cdr區(qū)形成了合理的抗原結(jié)合結(jié)構(gòu)域,為雌二醇小分子的結(jié)合提供了有利的空間構(gòu)象。對(duì)e2-vhh復(fù)合物進(jìn)行相互作用的分析時(shí)發(fā)現(xiàn),當(dāng)vhh的cdr區(qū),特別是cdr3區(qū)中存在氨基酸殘基含有氮元素時(shí),尤其是氨基中的氮元素,其可以通過(guò)孤對(duì)電子和雌二醇中位于結(jié)構(gòu)兩端的羥基氫形成氫鍵,另外當(dāng)cdr區(qū)含有芳香環(huán),并且和雌二醇中苯環(huán)結(jié)構(gòu)的相對(duì)空間位置合適時(shí),可以通過(guò)形成π-π鍵來(lái)加強(qiáng)雌二醇和vhh抗體的相互作用力。通過(guò)上述分析結(jié)果發(fā)現(xiàn),通過(guò)分子對(duì)接技術(shù),可以有效的完成抗體和雌二醇小分子之間的對(duì)接分析,實(shí)現(xiàn)了vhh虛擬抗體庫(kù)的虛擬篩選策略。此外通過(guò)利用超級(jí)計(jì)算機(jī),針對(duì)抗體庫(kù)的虛擬篩選,解決了其耗費(fèi)計(jì)算資源的問(wèn)題,實(shí)現(xiàn)了抗體庫(kù)中2000株vhh抗體的高效虛擬篩選。本篩選方法的建立利用了計(jì)算生物學(xué)中的分子對(duì)接技術(shù),并依托超級(jí)計(jì)算機(jī)實(shí)現(xiàn)了vhh虛擬抗體庫(kù)對(duì)雌二醇小分子的快速篩選,與傳統(tǒng)的抗體篩選技術(shù)相比,在對(duì)篩選平臺(tái)得到的結(jié)果進(jìn)行分析后,得到了雌二醇和vhh抗體之間相互作用模式等重要信息,為vhh抗體的親和力成熟以及抗體的設(shè)計(jì)等工作奠定了基礎(chǔ)。4.雌二醇vhh抗體的表達(dá)、純化與性質(zhì)鑒定選擇了常用的pet載體系列對(duì)vhh蛋白進(jìn)行了重組表達(dá)。首先,設(shè)計(jì)vhh的擴(kuò)增引物,在上游引物和下游引物中分別引入bamhi和ecori酶切位點(diǎn),為了保證酶切的效果,先將擴(kuò)增好的含有酶切位點(diǎn)的vhh基因片段克隆到pmd18-t質(zhì)粒中,然后對(duì)pmd18-t-vhh質(zhì)粒以及pet32a質(zhì)粒進(jìn)行雙酶切處理,通過(guò)t4ligase連接酶進(jìn)行連接,并轉(zhuǎn)化bl21(de3)感受態(tài)細(xì)胞,通過(guò)菌落pcr以及測(cè)序結(jié)果分析,選擇測(cè)序結(jié)果正確以及開放讀碼框正確的菌株進(jìn)行轉(zhuǎn)接培養(yǎng)并保存菌株。在進(jìn)行大量表達(dá)時(shí),首先將菌液進(jìn)行活化,然后轉(zhuǎn)接到大瓶培養(yǎng)基中,待培養(yǎng)菌液的od600值達(dá)到0.5-0.6之間,加入iptg進(jìn)行誘導(dǎo)表達(dá),37℃,12-15h過(guò)夜。通過(guò)sds-page檢測(cè)表達(dá)的菌液,vhh具有很高的表達(dá)量,通過(guò)超聲破碎后,VHH以包涵體的形式存在在破碎后離心的沉淀中,利用包涵變復(fù)性對(duì)VHH進(jìn)行了高純度的純化,并通過(guò)BCA蛋白定量試劑盒對(duì)純化后的可溶性VHH進(jìn)行蛋白定量。在雌二醇VHH抗體的性質(zhì)鑒定實(shí)驗(yàn)中,選擇了虛擬篩選結(jié)果中結(jié)合自由能值最低的一株抗體VHH165進(jìn)行驗(yàn)證,以間接ELISA和SPR兩個(gè)實(shí)驗(yàn)方法分別對(duì)VHH抗體的結(jié)合性質(zhì)、競(jìng)爭(zhēng)活性以及親和力常數(shù)進(jìn)行了測(cè)定。在雌二醇VHH抗體的間接ELISA實(shí)驗(yàn)研究中發(fā)現(xiàn),隨著VHH165的濃度增加,和包被在酶聯(lián)孔中的E2-OVA的結(jié)合逐漸增加,并且遠(yuǎn)高于VHH165和OVA的結(jié)合;通過(guò)間接ELISA初步驗(yàn)證,VHH165的IC50值為20.03ng/mL,最低檢測(cè)限LOD:IC10值為4.079ng/mL,通過(guò)SPR測(cè)定,VHH165的親和力為1.49mM。本研究應(yīng)用測(cè)序技術(shù)及計(jì)算生物學(xué)方法,建立了駱駝源天然單域抗體庫(kù),包括VHH實(shí)體庫(kù)以及VHH虛擬庫(kù),并利用分子對(duì)接的方法實(shí)現(xiàn)了雌二醇VHH抗體的快速虛擬篩選,在小分子與抗體的結(jié)合復(fù)合物的分析的基礎(chǔ)上獲得了相互作用模式的信息。此外,還建立了駱駝源單域抗體的高效原核表達(dá)體系,并檢測(cè)了雌二醇特異性抗體的結(jié)合性質(zhì)、競(jìng)爭(zhēng)活性以及親和力常數(shù),初步驗(yàn)證了該VHH抗體篩選平臺(tái)以及虛擬篩選策略。該VHH抗體篩選平臺(tái)和通過(guò)傳統(tǒng)方法建立的篩選方法相比有如下兩點(diǎn)優(yōu)勢(shì):一方面,該抗體庫(kù)利用了駱駝源的單域抗體,這類小型化的抗體具有分子量小,熱穩(wěn)定性高、可以耐受復(fù)雜應(yīng)用環(huán)境以及易制備等優(yōu)勢(shì),其有助于抗體制備時(shí)的基因操作、高水平表達(dá)表達(dá)以及易于保存等;另一方面,通過(guò)借助計(jì)算生物學(xué)方法,可以實(shí)現(xiàn)對(duì)抗體庫(kù)中的每一株抗體的遺傳背景、蛋白質(zhì)二級(jí)和三級(jí)結(jié)構(gòu)以及特異性抗體和識(shí)別靶標(biāo)相互作用模式的清晰認(rèn)識(shí)。在未來(lái)的工作中,通過(guò)綜合以上信息可以實(shí)現(xiàn)對(duì)特異性抗體的虛擬設(shè)計(jì),并大大提高了抗體制備后期親和力成熟的速度以及可靠性。在單域抗體庫(kù)建立后,通過(guò)本方法獲得的特異性VHH抗體,具有較好的親和力,以及較為全面的生物背景信息,可以在進(jìn)一步的抗體制備過(guò)程中開展可靠的親和力成熟及抗體設(shè)計(jì)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

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78;R155.5

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