1-4型登革病毒外膜蛋白基因DⅢ区的表达及其在血清学诊断和免疫保护方面的应用研究

发布时间：2018-05-26 09:03

本文选题：登革病毒 + 外膜蛋白　；参考：《福建医科大学》2007年博士论文

【摘要】： 近年来,随着登革热流行的日益严重,对登革病毒进行有效的防治是全世界共同关注的问题。本课题以登革病毒外膜基因DⅢ区(EDⅢ)为研究对象,进行诊断试剂盒的研制,同时构建了3种类型的登革疫苗进行免疫保护的研究。一、1-4型登革病毒外膜蛋白基因DⅢ区的表达及重组抗原在血清学诊断中的应用在大肠杆菌BL21(DE3)中分别克隆、表达1-4型登革病毒外膜蛋白基因DⅢ区,重组蛋白用电洗脱法进行纯化。纯化后的DEN1-4型重组蛋白分别应用于间接ELISA法检测相应型别DEN IgG抗体,以IFA为标准,其敏感性为100%;DEN1型重组蛋白应用于捕获ELISA法检测DEN1型感染者IgM抗体,不仅能检出所有IFA法IgM阳性的标本,而且还检出了2份IFA法未能检出的早期标本;DEN1型重组蛋白应用于夹心ELISA法检测DEN1型感染者IgM/IgG总抗体,其敏感性为82.6%;以IFA为标准,三种方法的特异性均为100%。纯化的重组蛋白混和后作为捕获抗原制成金标试剂条,应用于登革病毒感染者的检测,与PanBio公司的金标条检测结果相比,两者无显著性差别(P0.05)。二、登革疫苗的研制及免疫保护作用分析 1. 1-4型登革病毒重组亚单位疫苗的研制及免疫原性分析利用连接肽(Gly-Gly-Ser-Gly-Ser)3将DEN 1型与2型,3型与4型的EDⅢ基因片段连接在一起,构建了EDⅢ融合基因的原核表达载体,在大肠杆菌中表达融合重组蛋白。重组蛋白经高效液相色谱(HPLC)纯化后免疫BALB/c鼠,采用中和实验法测定血清DEN1-4型中和抗体效价,其滴度分别为1:34.9、1:45.3、1:24.7、1:38.4。免疫血清分别与1-4型的登革病毒作用后,攻击乳鼠,免疫血清对乳鼠保护率分别为100%、100%、83%、83%。 2.登革病毒EDⅢ融合基因真核表达载体的构建及DNA在小鼠中的免疫原性观察构建DEN1/2型、DEN3/4型EDⅢ融合基因的真核表达载体,用间接免疫荧光法和Western blot法检测重组真核表达载体在BHK-21细胞中的表达情况。结果表明,重组真核表达载体能在BHK-21细胞中表达目的蛋白。DNA质粒免疫BALB/c鼠,采用中和实验法测定血清DEN1-4中和抗体效价,其滴度分别为1:24.7、1:26.9、1:13.4、1:16.0。 3.EDⅢ融合蛋白及DNA质粒联合免疫小鼠诱生中和抗体的分析将DⅢ融合重组蛋白及含有EDⅢ融合基因的DNA质粒单独或联合免疫小鼠,观察其对小鼠的免疫原性,比较诱生中和抗体的能力。结果表明,联合免疫组产生的中和抗体效价显著高于重组蛋白免疫组及DNA免疫组,同时蛋白免疫组与DNA免疫组比较,中和抗体也表现为增高。 4.登革病毒EDⅢ区融合基因重组腺病毒载体的构建及鉴定采用腺病毒表达系统,构建DEN1/2型、DEN3/4型EDⅢ融合基因的重组腺病毒表达载体,并且在293A细胞系中包装成具有感染性的重组腺病毒,病毒滴度可达109PFU/ml。重组腺病毒感染哺乳动物细胞后,用间接免疫荧光法和Western blot法检测重组蛋白在细胞中的表达情况,结果显示,重组腺病毒感染哺乳动物细胞后能够表达目的蛋白。
[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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373

【引证文献】