本文選題：DTMUV　切入點：E　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鴨坦布蘇病毒(Duck Tembusu virus,DTMUV)是2010年初在我國的江浙地區(qū)新發(fā)的一種黃病毒,引起蛋鴨的產(chǎn)蛋量急劇下降甚至停止,給我國的養(yǎng)殖業(yè)造成嚴(yán)重的經(jīng)濟損失。近些年對其分離鑒定、基因組特征和診斷方法的報道較多,但是對于該病毒中和表位和受體的研究成果較少,尤其是針對DTMUV蛋白受體的研究至今仍無進展。囊膜E蛋白作為DTMUV主要的結(jié)構(gòu)蛋白,不僅可以刺激機體產(chǎn)生中和抗體,同時與受體結(jié)合介導(dǎo)病毒的入侵,對揭示病毒感染機制和免疫反應(yīng)至關(guān)重要。本研究克隆并表達了DTMUV E蛋白,制備了多株單克隆抗體,篩選出一株具有中和活性的單克隆抗體,通過鑒定發(fā)現(xiàn)其中和表位位于E蛋白的Domain I區(qū)域,這是首次在黃病毒E蛋白Domain I區(qū)域發(fā)現(xiàn)的中和表位,豐富了我們對黃病毒中和表位的認(rèn)識。通過對中和抗體的可變區(qū)進行克隆、序列分析和單鏈抗體(Sc Fv)的重組質(zhì)粒構(gòu)建,發(fā)現(xiàn)其輕鏈互補決定區(qū)序列具有顯著的特異性,純化的單鏈抗體具有一定的抗病毒效果,為針對該病的預(yù)防與治療奠定了基礎(chǔ)。采用免疫共沉淀和病毒-受體蛋白結(jié)合試驗(VOPBA)技術(shù)對DTMUV的受體進行了初步篩選,但是未檢測分離到與DTMUV易感細(xì)胞結(jié)合的蛋白受體,通過DTMUV E蛋白與血紅細(xì)胞的血凝試驗檢測發(fā)現(xiàn)E蛋白具有一定的血凝活性,提示該病毒受體可能屬于糖類,為該病的防控提供新的思路。具體研究內(nèi)容如下:1.DTMUV E單克隆抗體的制備與鑒定本研究根據(jù)實驗室分離的鴨坦布蘇病毒W(wǎng)H2014株E基因序列(Gen Bank KY235382),構(gòu)建了去信號肽成熟的E蛋白的重組質(zhì)粒p ET-28a-E,采用原核誘導(dǎo)表達和純化系統(tǒng)獲得高純度的E蛋白。將純化后的E蛋白免疫BALB/c小鼠制備針對E蛋白的多株單克隆抗體,通過間接ELISA、間接免疫熒光和Western blot篩選獲得2株穩(wěn)定分泌抗體的雜交瘤細(xì)胞株,分別命名為3B8G和3F12G。其腹水間接ELISA效價分別為1:51200和1:409600;亞類鑒定結(jié)果顯示,Mc Ab 3B8G重鏈為Ig G1類,Mc Ab 3F12G重鏈為Ig G3類,輕鏈均為κ鏈;間接免疫熒光和Western blot結(jié)果顯示,兩株單抗均能與過表達的E蛋白具有特異性結(jié)合反應(yīng),并且與DTMUV同樣產(chǎn)生特異性反應(yīng)。病毒中和實驗結(jié)果表明,Mc Ab 3B8G對DTMUV的中和效果較好。2.E蛋白中和抗原表位的鑒定通過生物信息學(xué)方法分析DTMUV E蛋白空間構(gòu)象,對E蛋白的三個結(jié)構(gòu)域進行截短突變體真核表達重組質(zhì)粒的構(gòu)建,間接ELISA、間接免疫熒光和Western blot檢測發(fā)現(xiàn)Mc Ab 3B8G識別E蛋白的DEI區(qū)域,Mc Ab 3F12G識別E蛋白的Domain II區(qū)域。為了進一步鑒定效果較好的中和抗體3B8G在E蛋白DEI區(qū)域內(nèi)的抗原表位最小功能區(qū),本研究通過構(gòu)建一系列覆蓋E蛋白Domain I全長的截短突變體真核表達重組質(zhì)粒,經(jīng)IFA和Western blot檢測,精確掃描定位了Mc Ab 3B8G識別的一個B細(xì)胞線性表位1FSCLGMQ7。3.抗DTMUV E蛋白單鏈抗體的制備與鑒定本研究在成功制備穩(wěn)定分泌抗DTMUV E蛋白單抗雜交瘤細(xì)胞株的基礎(chǔ)上,對兩株單抗的VL區(qū)域擴增,比對分析其序列發(fā)現(xiàn)兩株抗體在VL區(qū)域相似性較低,決定了抗體識別E蛋白結(jié)構(gòu)域的特異性。通過基因工程方法,拼接抗DTMUV E蛋白單鏈抗體基因并構(gòu)建了重組表達載體p ET 28a-DTMUV-E Sc Fv,表達純化得到的單鏈抗體具有良好的抗病毒活性。4.DTMUV E蛋白受體的篩選為了研究DTMUV在易感細(xì)胞上的受體蛋白分子,本研究表達純化了E蛋白和Domain III蛋白,通過間接免疫熒光法篩選與DTMUV易感細(xì)胞穩(wěn)定結(jié)合的蛋白,發(fā)現(xiàn)E蛋白能夠穩(wěn)定結(jié)合DEF細(xì)胞、DF1細(xì)胞和Vero細(xì)胞。然后采用膜蛋白提取試劑盒提取以上三種細(xì)胞的膜蛋白,將膜蛋白、純化的E蛋白和抗DTMUV的單抗進行免疫共沉淀和病毒-蛋白結(jié)合篩選,結(jié)果均未篩選到特異性可分離的DTMUV蛋白受體,血凝試驗提示鴨坦布蘇病毒的受體可能為糖類,為進一步研究病毒受體提供了思路。綜上所述,本研究鑒定出DTMUV位于E蛋白Domain I結(jié)構(gòu)域內(nèi)的中和表位;分析了兩株識別不同抗原區(qū)的單克隆抗體輕鏈可變區(qū)序列差異,構(gòu)建了抗DTMUV E蛋白的單鏈抗體融合表達載體;對DTMUV的蛋白受體分子進行了初步篩選。本研究對建立鴨坦布蘇病毒的防控與治療技術(shù)具有重要意義。
[Abstract]:Duck Tembusu virus (Duck Tembusu, virus, DTMUV) is a kind of flavivirus in early 2010 China's Jiangsu and Zhejiang new hair, caused by the sharp decline in duck egg production or even stop, causing serious economic losses to the aquaculture of our country in recent years. The isolation and identification, characteristics and diagnostic methods of the genome are reported, but research on the virus neutralizing epitopes and receptor are few, especially on DTMUV receptor is still no progress. Envelope E protein as the major structural protein of DTMUV, not only can stimulate the organism to produce neutralizing antibody, combined with virus and receptor mediated invasion, to reveal the mechanism of viral infection and the immune response is crucial the study on cloning and expression of DTMUV E protein, polyclonal monoclonal antibodies were prepared and screened a strain of monoclonal antibodies with neutralizing activity, through the identification of the epitopes found a In the Domain I region of E protein, which was first discovered in the Domain I region of flavivirus E protein epitopes, to enrich our understanding of virus neutralizing epitopes were cloned. Through variable region of neutralizing antibody, and sequence analysis of scFv (Sc Fv) to construct the recombinant plasmid sequence has found out. The remarkable specificity of light chain complementarity, the purified scFv has certain antiviral effect, for the prevention and treatment of the disease of the foundation. By immunoprecipitation and virus - receptor protein binding assay (VOPBA) technique on DTMUV by the initial screening, but not easy to separation and DTMUV detection sense of cell binding protein receptor, by hemagglutination test for the detection of DTMUV E protein and red blood cells showed that E protein has hemagglutination activity, suggesting that the virus receptor may belong to carbohydrate, provide for disease prevention and control of new Ideas. The specific contents are as follows: preparation and identification on the basis of duck Tembusu virus WH2014 strain E gene sequence of 1.DTMUV E isolated monoclonal antibody (Gen Bank KY235382), to construct the signal peptide of mature E protein recombinant plasmid P ET-28a-E, induced by prokaryotic expression and purification system of high purity the E mice were immunized with BALB/c protein. The purified E protein to prepare monoclonal antibodies to E protein, indirect ELISA, indirect immunofluorescence and Western blot screened 2 strains of stable antibody secreting hybridoma cell lines, named 3B8G and 3F12G. in the ascites titer of ELISA were 1:51200 and 1:409600; subtype identification showed that Mc Ab 3B8G heavy chain Ig G1, Mc Ab 3F12G heavy chain Ig G3, were light chain kappa chain; indirect immunofluorescence and Western blot results showed that the two McAbs with overexpression of E The protein has a specific binding reaction, and DTMUV also produced specific virus neutralizing reaction. The experimental results show that the identification of Mc Ab 3B8G of DTMUV and.2.E protein and better antigen through bioinformatics analysis of DTMUV on the conformation of E protein, E egg three domain of truncated mutant really nuclear construction, expression recombinant plasmid of indirect ELISA, immunofluorescence and Western blot detected DEI Mc Ab 3B8G E region recognition protein, Domain II Mc Ab 3F12G E protein region recognition. In order to further identification of neutralizing antibody 3B8G good effect in E protein DEI region epitope minimum functional areas, this study through the construction of a series of E Domain I protein covering full-length truncated mutant recombinant eukaryotic expression plasmid, the IFA and Western blot detection, Mc Ab 3B8G scanning and positioning accurate identification of a B cell Preparation and identification of the research in the successful preparation of E protein stably secreting monoclonal antibody against DTMUV linear epitopes on 1FSCLGMQ7.3. protein of single chain antibody against DTMUV E, VL region of two strains of monoclonal antibody amplification, the sequence alignment analysis showed that two strains of antibodies in the VL region of low similarity, determines the specific antibody recognition of E protein domains. By gene engineering method, protein splicing of single chain antibody against DTMUV E gene and construct the recombinant expression vector p ET 28a-DTMUV-E Sc Fv, the expression of purified scFv obtained with antiviral activity of E protein.4.DTMUV receptor of DTMUV in order to study the receptor protein in susceptible cells. The study on the expression and purification of E protein and Domain III protein by indirect immunofluorescence assay and screening of DTMUV susceptible cell stable binding protein, E protein can stably bind to DEF cells, DF1 Cell membrane protein and Vero cells. Then the membrane protein extraction kit to extract more than three kinds of cells, the membrane protein, the purified E protein and anti DTMUV monoclonal antibody immunoprecipitation and virus protein binding screening, the results were not screened for specific separation of DTMUV protein by hemagglutination test indicated that the body. Duck Tembusu virus receptor may be sugar, provides ideas for further study of virus receptor. In conclusion, this study identified DTMUV in E protein Domain I domain in neutralizing epitope; analysis of antibody light chain variable region sequence difference of two strains of monoclonal antigen recognition in different areas, construction of anti DTMUV scFv antibody E fusion protein expression vector; protein receptor molecules on DTMUV were screened. Has important significance for prevention and treatment technology for the establishment of duck Tembusu virus in this study.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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