本文選題：馬爾堡出血熱 + 馬爾堡病毒�。� 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：馬爾堡出血熱(Marburg Hemorrhagic Fever,MHF)也稱為馬爾堡病毒病,是由馬爾堡病毒(Marburg virus,MARV)引起的嚴(yán)重出血熱的人獸共患傳染病,發(fā)病后幾乎高達(dá)90%的死亡率,至今仍無商品化的治療藥物和疫苗。該病毒首次在德國的馬爾堡城市的實驗室內(nèi)分離并因此被命名。大多數(shù)嚴(yán)重的病例主要發(fā)生在醫(yī)療設(shè)備相對落后的非洲。馬爾堡出血熱主要通過密切接觸傳播,因此應(yīng)該對病人及可疑病例進(jìn)行隔離,建立相關(guān)的快速安全的檢測方法是控制該病傳播的最基本的手段。MARV的基因組由單股負(fù)鏈RNA構(gòu)成,病毒基因組全長約19.1kb,共包括7個閱讀框(ORF),分別編碼7種蛋白:核蛋白(NP)、包膜糖蛋白(GP)、結(jié)構(gòu)蛋白(VP30和VP35)、基質(zhì)蛋白VP24和VP40、RNA依賴的RNA多聚酶(L蛋白)。該病毒目前只發(fā)現(xiàn)一個血清型。其中,MARV糖蛋白(Glycoprotein,GP)是病毒僅有的表面蛋白,能夠誘導(dǎo)機(jī)體產(chǎn)生中和抗體。GP由GP1和GP2構(gòu)成,受體結(jié)合域(RBD,aa38-188)是位于GP1內(nèi)N端的一個不連續(xù)的獨立折疊區(qū)域,能夠比完整的GP1更好的與細(xì)胞受體結(jié)合產(chǎn)生反應(yīng)。本研究利用大腸桿菌原核表達(dá)系統(tǒng)表達(dá)重組MARV-GP-RBD蛋白,并以該蛋白為基礎(chǔ)建立MARV抗體間接ELISA方法并制備抗MARV-GP-RBD蛋白的單克隆抗體及多克隆抗體;建立了馬爾堡病毒樣顆粒糖蛋白含量檢測雙抗體夾心ELISA方法。原核表達(dá)馬爾堡病毒糖蛋白受體結(jié)合域及純化并制備單克隆抗體及多克隆抗體。目的原核表達(dá)并純化馬爾堡病毒(Maburg Virus,MARV)糖蛋白(Glycoprotein,GP)的受體結(jié)合域(Receptor Binding Domain,RBD)蛋白,以之為抗原免疫家兔制備多克隆抗體,免疫小鼠制備單克隆抗體。方法參照GenBank提供的MARV GP全基因序列,找到主要抗原表位區(qū)域(Receptor Binding Domain,RBD),設(shè)計特異性引物,采用PCR方法擴(kuò)增RBD基因,擴(kuò)增產(chǎn)物經(jīng)雙酶切(EcoRⅠ/XhoⅠ)后定向克隆至原核表達(dá)載體pET-30a(+),構(gòu)建重組表達(dá)質(zhì)粒pET-30a(+)-GP-RBD,轉(zhuǎn)化BL21(DE3)感受態(tài)表達(dá)宿主菌,在不同條件下(時間、IPTG濃度、溫度)誘導(dǎo)表達(dá)目的蛋白,并用His-Band N+柱進(jìn)行親和層析純化;以純化的重組pET-30a(+)-GP-RBD蛋白免疫家兔和小鼠,制備多克隆抗體和單克隆抗體。通過SDS-PAGE、Western blot和IFA鑒定目的蛋白的反應(yīng)原性和免疫原性。結(jié)果PCR擴(kuò)增到長度為453bp的RBD基因片段;構(gòu)建的重組質(zhì)粒pET-30a(+)-GP-RBD經(jīng)雙酶切后得到與目的片段長度相同的特異性條帶,測序結(jié)果顯示沒有突變;轉(zhuǎn)化產(chǎn)物在培養(yǎng)7h、終濃度為0.4mmol/l IPTG和37℃條件下能夠充分誘導(dǎo)目的蛋白表達(dá),得到分子質(zhì)量大小為25ku的重組蛋白,主要以包涵體形式存在,BCA試劑盒產(chǎn)量測定,每升誘導(dǎo)的重組菌可純化約20mg左右純度較高的目的蛋白;Western blot檢測證實了重組pET-30a(+)-GP-RBD蛋白能同時被抗His標(biāo)簽的單抗、兔源多抗和鼠源單抗識別并發(fā)生特異性反應(yīng),證明該蛋白具有較好的反應(yīng)原性;IFA鑒定證實所制備的兔源多抗和鼠源單抗均能夠特異性識別表達(dá)MARV GP蛋白的重組桿狀病毒rBacmid-GP-VP40,證明重組蛋白具有良好的免疫原性。結(jié)論成功表達(dá)純化了MARV GP蛋白RBD蛋白,并完成了兔源多抗和鼠源單抗的制備與鑒定,為MARV亞單位疫苗的制備和抗原、抗體檢測方法的建立奠定了基礎(chǔ)。間接ELISA檢測馬爾堡病毒抗體方法的建立及初步應(yīng)用。目的建立MARV抗體間接ELISA檢測方法,并對所建立方法的相關(guān)條件進(jìn)行了優(yōu)化。方法以純化后的MARV糖蛋白的受體結(jié)合域蛋白作為包被抗原,MARV病毒樣顆粒免疫馬匹獲得的高免馬血清為一抗,MARV陰性馬血清為陰性對照,HRP標(biāo)記山羊抗馬IgG為二抗,優(yōu)化反應(yīng)條件并評價其特異性和敏感性。結(jié)果抗原的最佳包被質(zhì)量濃度為4ug/ml;血清最佳稀釋度為1:640;酶標(biāo)二抗的最佳濃度為1:5000;優(yōu)化后的間接ELISA方法可特異性檢測MARV抗體,與埃博拉病毒,西尼羅河熱病毒,委內(nèi)瑞拉馬腦炎,裂谷熱等病毒的陽性血清沒有交叉反應(yīng)。批內(nèi)、批間試驗變異系數(shù)均小于10%。結(jié)論本研究成功建立了MARV抗體檢測間接ELISA方法,為MARV的血清學(xué)調(diào)查奠定基礎(chǔ)。雙抗體夾心ELISA定量檢測馬爾堡病毒糖蛋白檢測方法的建立及初步應(yīng)用。目的定量檢測馬爾堡病毒病毒樣顆粒糖蛋白含量。方法以純化的MARV GP A21單克隆抗體為捕獲抗體,純化的A1單抗為檢測抗體,純化的原核表達(dá)的MARV GP作為標(biāo)準(zhǔn)品繪制標(biāo)準(zhǔn)曲線。優(yōu)化并選擇最佳作用條件并對建立的方法進(jìn)行評價。結(jié)果A21抗體最佳稀釋度為2560(包被濃度為0.742ug/ml),HRP標(biāo)記后的A1最佳稀釋度為1:8000。與埃博拉病毒,西尼羅河熱病毒,裂谷熱病毒,中東呼吸綜合征病毒等無交叉反應(yīng),批內(nèi)、批間試驗變異系數(shù)均小于10%,對MARV的檢測靈敏度達(dá)21.052 ng/ml。結(jié)論該方法可特異性定量檢測MARV,與其他病毒均不發(fā)生反應(yīng)。該方法靈敏度、特異性高、重復(fù)性好。該方法可對馬爾堡病毒樣顆粒中的糖蛋白的相對含量進(jìn)行監(jiān)測。該方法適用于馬爾堡病毒樣顆粒疫苗免疫劑量的測定。
[Abstract]:Marburg Hemorrhagic Fever (MHF), also known as Marburg virus disease, is a human zoonosis of severe hemorrhagic fever caused by the Marburg virus (MARV), almost 90% of mortality after the onset of the disease, and there is still no commercialized treatment and vaccine. The virus was first in the Marburg city of Germany. Most serious cases mainly occur in relatively backward Africa. The Marburg haemorrhagic fever is mainly transmitted through close contact and therefore should be isolated from patients and suspected cases, and the establishment of a related rapid and safe method of detection is the most basic means of controlling the transmission of the disease,.MARV. The genome is composed of single strand negative chain RNA. The total length of the virus genome is about 19.1kb, including 7 reading frames (ORF), which encode 7 proteins: nucleoprotein (NP), membrane glycoprotein (GP), structural protein (VP30 and VP35), matrix protein VP24 and VP40, RNA dependent RNA polyenzyme (L protein). The virus currently only found one serotype. Oprotein, GP) is the only surface protein of the virus, which can induce the organism to produce neutralizing antibody.GP, which is composed of GP1 and GP2. The receptor binding domain (RBD, aa38-188) is an independent, discontinuous folding region located at the N end of GP1, which can better react with the cell receptor than the complete GP1. This study uses the Escherichia coli prokaryotic expression system. The recombinant MARV-GP-RBD protein was expressed and the indirect ELISA method of MARV antibody was established on the basis of the protein, and the monoclonal antibody and polyclonal antibody against MARV-GP-RBD protein were prepared. A double antibody sandwich ELISA method was established for the detection of Marburg virus like granular glycoprotein content. The binding domain of the glycoprotein receptor of Marburg virus was expressed and purified. Preparation of monoclonal antibodies and polyclonal antibodies. Aim prokaryotic expression and purification of the receptor binding domain (Receptor Binding Domain, RBD) of Maburg Virus (MARV) glycoprotein (Glycoprotein, GP). The polyclonal antibody was prepared for antigen immunization in rabbits, and monoclonal antibodies were prepared from the immunized mice. The method referred to MARV GP provided by GenBank. The whole gene sequence, Receptor Binding Domain (RBD) was found, specific primers were designed, RBD gene was amplified by PCR, and the amplified products were cloned to the prokaryotic expression vector pET-30a (+) after double enzyme digestion (EcoR I /Xho I), and the recombinant plasmid was constructed to plasmid pET-30a (+) -GP-RBD, and the host bacteria were transformed into the receptive state of the BL21. The target protein was induced under different conditions (time, IPTG concentration, temperature) and purified by affinity chromatography with His-Band N+ column, and the purified recombinant pET-30a (+) -GP-RBD protein was used to immunize rabbits and mice to prepare polyclonal and monoclonal antibodies. The reactivity and immunogenicity of the target protein were identified by SDS-PAGE, Western blot and IFA. Results PCR amplified the RBD gene fragment length of 453bp, and the recombinant plasmid pET-30a (+) -GP-RBD obtained the same specific bands as the length of the target fragment after double enzyme digestion. The sequencing results showed no mutation, and the transformed product could fully induce the expression of the target protein in the culture of 7h, the final concentration of 0.4mmol/l IPTG and 37 C. The recombinant protein with a molecular weight of 25ku, mainly in inclusion body form, determination of BCA kit yield, and purified recombinant bacteria with high purity about 20mg about 20mg per liter, and Western blot detection confirmed that the recombinant pET-30a (+) -GP-RBD protein can be simultaneously identified with anti His labelling monoclonal antibody, rabbit source polyclonal and mouse monoclonal antibody recognition and The specific reaction showed that the protein had good reactivity. The IFA identification confirmed that the rabbit source polyclonal and mouse McAbs were able to identify the recombinant baculovirus rBacmid-GP-VP40 specifically expressing MARV GP protein, which proved that the recombinant protein had good immunogenicity. The MARV GP protein RBD protein was successfully expressed and purified. The preparation and identification of rabbit source polyclonal and mouse monoclonal antibody were completed. The basis was laid for the preparation and antigen of MARV subunit vaccine and the establishment of antibody detection methods. The establishment and preliminary application of the indirect ELISA method for detecting the antibody of Marburg virus was established. Objective to establish the indirect ELISA detection method for MARV antibody, and to carry out the related conditions of the established method. Methods using the purified MARV glycoprotein receptor binding domain protein as the inclusion antigen, the high free horse serum obtained by the MARV virus like particles, the MARV negative horse serum as the negative control, the HRP labeled Goat anti horse IgG as two, the optimum reaction conditions and the evaluation of its specificity and sensitivity. The mass concentration was 4ug/ml, the best dilution of serum was 1:640, the best concentration of anti enzyme two was 1:5000, and the optimized indirect ELISA method was specific to detect MARV antibody. There was no cross reaction with Ebola virus, West Nile heat virus, Venezuela horse encephalitis, rift heat and so on. The coefficient of variation in batch test was small. In 10%. conclusion, the indirect ELISA method for the detection of MARV antibody was established successfully, which laid the foundation for the serological investigation of MARV. The quantitative detection of Marburg virus like glycoprotein content by double antibody sandwich ELISA quantitative detection of Marburg virus glycoprotein. Method for the purification of the purified MARV GP A21 monomer. The cloned antibody was captured. The purified A1 monoclonal antibody was the detection antibody and the purified prokaryotic expression MARV GP was used as standard curve. The optimum conditions were optimized and selected to evaluate the established method. The best dilution degree of A21 antibody was 2560 (the concentration was 0.742ug/ml), and the best dilution degree of A1 after HRP was 1:8000. There was no cross reaction with Ebola virus, West Nile heat virus, Rift Valley fever virus, Middle East respiratory syndrome virus and so on. In batch, the coefficient of variation was less than 10% in batch test, and the sensitivity of MARV was 21.052 ng/ml.. Conclusion the method can detect MARV with specificity and no reaction to his virus. The sensitivity and specificity of this method is high. The method can be used to monitor the relative content of glycoproteins in Marburg virus like particles. This method is suitable for the determination of the immune dose of Marburg virus like particle vaccine.

【學(xué)位授予單位】：吉林農(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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相關(guān)期刊論文 前10條

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本文編號：1777552