發(fā)布時(shí)間：2018-05-26 09:03

  本文選題：登革病毒 + 外膜蛋白��； 參考：《福建醫(yī)科大學(xué)》2007年博士論文

【摘要】： 近年來(lái),隨著登革熱流行的日益嚴(yán)重,對(duì)登革病毒進(jìn)行有效的防治是全世界共同關(guān)注的問(wèn)題。本課題以登革病毒外膜基因DⅢ區(qū)(EDⅢ)為研究對(duì)象,進(jìn)行診斷試劑盒的研制,同時(shí)構(gòu)建了3種類(lèi)型的登革疫苗進(jìn)行免疫保護(hù)的研究。一、1-4型登革病毒外膜蛋白基因DⅢ區(qū)的表達(dá)及重組抗原在血清學(xué)診斷中的應(yīng)用 在大腸桿菌BL21(DE3)中分別克隆、表達(dá)1-4型登革病毒外膜蛋白基因DⅢ區(qū),重組蛋白用電洗脫法進(jìn)行純化。純化后的DEN1-4型重組蛋白分別應(yīng)用于間接ELISA法檢測(cè)相應(yīng)型別DEN IgG抗體,以IFA為標(biāo)準(zhǔn),其敏感性為100%;DEN1型重組蛋白應(yīng)用于捕獲ELISA法檢測(cè)DEN1型感染者IgM抗體,不僅能檢出所有IFA法IgM陽(yáng)性的標(biāo)本,而且還檢出了2份IFA法未能檢出的早期標(biāo)本;DEN1型重組蛋白應(yīng)用于夾心ELISA法檢測(cè)DEN1型感染者IgM/IgG總抗體,其敏感性為82.6%;以IFA為標(biāo)準(zhǔn),三種方法的特異性均為100%。純化的重組蛋白混和后作為捕獲抗原制成金標(biāo)試劑條,應(yīng)用于登革病毒感染者的檢測(cè),與PanBio公司的金標(biāo)條檢測(cè)結(jié)果相比,兩者無(wú)顯著性差別(P0.05)。 二、登革疫苗的研制及免疫保護(hù)作用分析 1. 1-4型登革病毒重組亞單位疫苗的研制及免疫原性分析利用連接肽(Gly-Gly-Ser-Gly-Ser)3將DEN 1型與2型,3型與4型的EDⅢ基因片段連接在一起,構(gòu)建了EDⅢ融合基因的原核表達(dá)載體,在大腸桿菌中表達(dá)融合重組蛋白。重組蛋白經(jīng)高效液相色譜(HPLC)純化后免疫BALB/c鼠,采用中和實(shí)驗(yàn)法測(cè)定血清DEN1-4型中和抗體效價(jià),其滴度分別為1:34.9、1:45.3、1:24.7、1:38.4。免疫血清分別與1-4型的登革病毒作用后,攻擊乳鼠,免疫血清對(duì)乳鼠保護(hù)率分別為100%、100%、83%、83%。 2.登革病毒EDⅢ融合基因真核表達(dá)載體的構(gòu)建及DNA在小鼠中的免疫原性觀察 構(gòu)建DEN1/2型、DEN3/4型EDⅢ融合基因的真核表達(dá)載體,用間接免疫熒光法和Western blot法檢測(cè)重組真核表達(dá)載體在BHK-21細(xì)胞中的表達(dá)情況。結(jié)果表明,重組真核表達(dá)載體能在BHK-21細(xì)胞中表達(dá)目的蛋白。DNA質(zhì)粒免疫BALB/c鼠,采用中和實(shí)驗(yàn)法測(cè)定血清DEN1-4中和抗體效價(jià),其滴度分別為1:24.7、1:26.9、1:13.4、1:16.0。 3.EDⅢ融合蛋白及DNA質(zhì)粒聯(lián)合免疫小鼠誘生中和抗體的分析 將DⅢ融合重組蛋白及含有EDⅢ融合基因的DNA質(zhì)粒單獨(dú)或聯(lián)合免疫小鼠,觀察其對(duì)小鼠的免疫原性,比較誘生中和抗體的能力。結(jié)果表明,聯(lián)合免疫組產(chǎn)生的中和抗體效價(jià)顯著高于重組蛋白免疫組及DNA免疫組,同時(shí)蛋白免疫組與DNA免疫組比較,中和抗體也表現(xiàn)為增高。 4.登革病毒EDⅢ區(qū)融合基因重組腺病毒載體的構(gòu)建及鑒定 采用腺病毒表達(dá)系統(tǒng),構(gòu)建DEN1/2型、DEN3/4型EDⅢ融合基因的重組腺病毒表達(dá)載體,并且在293A細(xì)胞系中包裝成具有感染性的重組腺病毒,病毒滴度可達(dá)109PFU/ml。重組腺病毒感染哺乳動(dòng)物細(xì)胞后,用間接免疫熒光法和Western blot法檢測(cè)重組蛋白在細(xì)胞中的表達(dá)情況,結(jié)果顯示,重組腺病毒感染哺乳動(dòng)物細(xì)胞后能夠表達(dá)目的蛋白。
[Abstract]:In recent years, with the increasingly serious epidemic of dengue fever, the effective prevention and control of dengue virus is a common concern in the world. This subject takes the dengue virus outer membrane gene D III (ED III) as the research object, develops the diagnostic kit, and constructs 3 types of dengue vaccine for immunization protection. 1, type 1-4 dengue Expression and recombinant antigen of outer membrane protein gene D III region and its application in serological diagnosis
The 1-4 dengue virus outer membrane protein gene D III was cloned respectively in the Escherichia coli BL21 (DE3). The recombinant protein was purified by electroelution. The purified recombinant protein was applied to the indirect ELISA method to detect the corresponding DEN IgG antibody, which was based on IFA as the standard, and its sensitivity was 100%, and the DEN1 type recombinant protein was applied to capture ELISA. The detection of IgM antibody of DEN1 type infected persons was not only detected in all IFA IgM positive specimens, but also in 2 early specimens which were not detected by IFA. The DEN1 recombinant protein was used to detect the total IgM/IgG antibody of DEN1 type infected persons with the sandwich ELISA method, and its sensitivity was 82.6%. The specificity of the three methods was 100%. purified with IFA as the standard. The recombinant protein was used as the capture antigen to make the gold labeling reagent, which was applied to the detection of dengue virus infection, and there was no significant difference compared with the gold mark test results of PanBio company (P0.05).
Two, the development and immune protection of dengue vaccine.
The development and immunogenicity of the recombinant subunit vaccine of type 1. 1-4 dengue virus (dengue virus), using Gly-Gly-Ser-Gly-Ser 3 to connect DEN 1 with type 2, 3 and 4 type ED III gene fragments, the prokaryotic expression vector of ED III fusion gene was constructed and the recombinant protein was fused in Escherichia coli. The recombinant protein was determined by high performance liquid chromatography (HPLC) the purified BALB/c mice were immunized and the neutralization antibody titer of serum DEN1-4 type was measured by neutralization test. The titer of the immunized mice was 100%, 100%, 83%, 83%., respectively, after the action of the 1:34.9,1:45.3,1:24.7,1:38.4. immune sera and type 1-4 dengue virus respectively.
Construction of 2. dengue virus ED III fusion gene eukaryotic expression vector and immunogenicity of DNA in mice
The eukaryotic expression vector of DEN1/2 type, DEN3/4 type ED III fusion gene was constructed. The expression of recombinant eukaryotic expression vector in BHK-21 cells was detected by indirect immunofluorescence and Western blot. The results showed that the recombinant eukaryotic expression vector could express the target protein.DNA plasmid in BHK-21 cells to immune BALB/c mice, and the neutralization test was used to determine the expression of the eukaryotic expression vector in BHK-21 cells. The titers of serum DEN1-4 neutralizing antibodies were 1:24.7,1:26.9,1:13.4,1:16.0.
Analysis of induced neutralization antibody in mice immunized with 3.ED III fusion protein and DNA plasmid
The immunogenicity of D III fusion recombinant protein and the DNA plasmid containing ED III fusion gene were observed in mice. The results showed that the neutralization antibody titer produced by the combined immunization group was significantly higher than that of the recombinant protein immunization group and the DNA immune group, and the protein immunization group and the DNA immunization group were also compared. In comparison, neutralizing antibodies also increased.
Construction and identification of recombinant adenovirus vector carrying 4. ED fusion gene of dengue virus
The recombinant adenovirus expression vector of DEN1/2 type, DEN3/4 type ED III fusion gene was constructed by adenovirus expression system, and an infectious recombinant adenovirus was packaged in the 293A cell line. The virus titer could reach the 109PFU/ml. recombinant adenovirus infected mammalian cells, and the recombinant eggs were detected by indirect immunofluorescence and Western blot method. The expression of white cells in cells showed that recombinant adenovirus could express target proteins after infection with mammalian cells.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R373

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王金章;翁育偉;嚴(yán)延生;;登革Ⅱ型病毒E蛋白DⅢ區(qū)的表達(dá)及其免疫反應(yīng)性鑒定[J];中國(guó)病毒病雜志;2013年01期

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本文編號(hào)：1936649