甲型流感病毒亚型间血清学交叉反应特征研究及反向遗传系统的优化
发布时间：2018-11-01 19:48
【摘要】：甲型流感病毒严重危害人类健康,由于其变异引发的大流行对人类造成严重威胁,近年来人感染禽流感病毒(H5N1)和甲型H1N1流感的流行,进一步提示加强甲型流感病毒的研究和控制的重要性。 血凝素(HA)和神经氨酸酶(NA)是甲型流感病毒编码的糖蛋白,变异性强,根据抗原性不同可进一步分为16个HA亚型(H1-H16)和9个NA亚型(N1-N9)。尽管HA和NA是流感病毒主要的免疫原,但由于HA和NA的变异性强,亚型众多,而目前甲型流感病毒16个HA和9个NA不同亚型之间的免疫交叉关系尚未系统研究,对流感病毒亚型特异性免疫检测技术的建立和疫苗的研发提出了严峻的考验。为此,本研究对甲型流感病毒16个亚型的HAl和9个亚型NA的交叉反应性进行了研究,以期阐明其免疫反应特征,为甲型流感病毒免疫检测技术和通用疫苗的研制提供可行性的理论依据。另一方面,近年来反向遗传系统的发明为人工改造甲型流感病毒和研发疫苗提供了关键技术平台,但现有系统的拯救效率有待提高,本研究拟通过对甲型流流感病毒反向遗传系统的转录／表达载体的改建提高其拯救效率。 一、甲型流感病毒HA和NA血清学交叉反应特征研究 对本实验选取的甲型流感病毒16个亚型的HA基因和9个亚型的NA基因的序列进行了分析,结果表明,16个HA和9个NA的核苷酸与氨基酸序列的进化树都主要向两个趋势进化,16个HA核苷酸序列之间的同源率在28%-76.7%之间,其氨基酸序列之间的同源率在36.2%-78.5%之间,9个NA亚型核苷酸序列之间的同源率在43.1%-70.4%之间,其氨基酸序列之间的同源率在37.9%66.6%之间。尽管16个HA和9个NA基因的预测B细胞线性表位分布区域一致,但其具体表位组成却存在差异,这些信息为进行各亚型之间的血清学交叉反应以及确定亚型特异性表位序列提供研究方向,进而为研究HA和NA的结构与功能、免疫识别、构建HA和NA的突变体以及选择表达新型HA和NA分子、研发诊断和基因工程疫苗奠定了基础。 随后将PCR扩增的经密码子优化的16个HA1和9个NA基因克隆到改建的gp67-pFastBac1载体中,获得了25个重组杆状病毒。将重组杆状病毒分别感染Sf9细胞,分别收取上清和细胞用抗6×His抗体进行Western blot分析。结果表明,16个HA1和9个NA基因的表达产物分子质量分别约为52.5kD与71.6kD,与预期相符,并且16个HA1和9个NA基因还呈分泌性表达。其中H5-HA1和H9-HA1经Ni柱纯化后的纯度达90%左右,回收率分别为0.5%和0.9%。同时,将16个亚型的HA和9个亚型的NA基因克隆到5型重组腺病毒载体中,得到了25个重组腺病毒。分别将25个重组腺病毒通过滴鼻途径免疫BALB/c小鼠,制备了相应亚型的抗体,三次免疫后ELISA检测阳转率为100%,ELISA检测的抗体效价约为1：6400左右。利用杆状病毒系统表达的各个亚型的目的蛋白作为抗原,利用重组腺病毒免疫BALB/c小鼠制备的血清作为抗体,通过ELISA方法确定16个HA亚型及9个NA亚型之间的血清学交叉反应关系。结果表明,HAl和NA各亚型之间的交叉反应关系错综复杂,H2、H5、H7亚型与其他亚型之间的交叉反应弱,而H6、H12、H16与其他亚型之间的交叉反应强。N1、N2亚型与其他亚型之间的交叉反应强,HA1和NA各亚型之间的交叉反应结果与其序列分析的结果存在一定的差异。 二、甲型流感病毒反向遗传系统的优化 利用RT-PCR的方法扩增了甲型流感病毒A/PR/8/34(H1N1)毒株8个基因片断,克隆入pIVVII载体中,其中在PB1片段中引入了3个沉默突变标签。将8个功能质粒共转染293T和MDCK混合培养细胞,利用细胞病变、RT-PCR、电镜技术等证明该系统可成功拯救甲型流感病毒。为提高甲型流感病毒的拯救效率,用人工合成的SCPI启动子[1]插入PIVVII载体,构建出pIVVS质粒,再将A/PR/8/34(H1N1)的8个基因片断克隆入pIVVS载体中,把优化后的8个功能质粒共转染293T和MDCK混合细胞,在共转染后的24h、48h、72h及96h分别收获转染细胞的上清和细胞,通过EGFP表达量、NP表达的时相变化以及血凝试验鉴定两个包装系统的拯救效率。结果表明,优化后系统不同时间点的EGFP表达量、NP表达量及血凝效价均高于未优化系统,说明利用高强度的SCPI启动子可有效提高甲型流感病毒的包装效率,为改进甲型流感病毒疫苗的研发系统奠定了基础。
[Abstract]:Influenza A virus is a serious threat to human health, which poses a serious threat to human beings due to the pandemic of influenza A virus. In recent years, people are infected with avian influenza virus (H5N1) and influenza A (H1N1) influenza, and further suggests the importance of strengthening the research and control of influenza A virus. Hemagglutinin (HA) and neuraminase (NA) are the glycoprotein encoded by influenza A virus, which can be further divided into 16 HA subtypes (H1-H16) and 9 NA subtypes according to different antigenicity (N1-N 9). Although HA and NA are the main immunogens of influenza virus, because of the strong variability of HA and NA, the subtypes are numerous, and the immune cross-relationship between the 16 HA and 9 NA subtypes of influenza A virus has not yet been completed. The establishment of specific immune detection technology for influenza virus subtypes and the research and development of vaccine have been put forward. In this study, the cross reactivity of HAl and 9 subtypes of influenza A virus was studied in order to elucidate its immune response characteristics, and to provide a feasible theory for the development of influenza A virus detection technology and universal vaccine. According to the invention, in recent years, the invention of the reverse genetic system provides a key technology platform for artificially modifying the influenza A virus and the R & D vaccine, but the rescue efficiency of the existing system needs to be This study is intended to improve its rescue by modifying the transcription/ expression vector of influenza A virus reverse genetic system. Efficiency. First, Influenza A HA and NA Serological Cross Response Characteristics: A Study of the HA Gene of 16 Subtypes of Influenza A Virus and the NA Gene of 9 Subtypes of Influenza A Virus The results showed that the homology of 16 HA and 9 NA sequences with amino acid sequence was mainly two trend evolutions, and the homology among 16 HA sequences was 28%. Between-76.7%, the homology between the amino acid sequences of the 9 NA subtypes ranged from 43.1% to 70.4%, and the homology between the amino acid sequences was 37. 9. While 16 HA and 9 NA genes predict B cell linear table bit distribution, there are differences in specific table bits, which are serologic cross reactions between subtypes and determine subtype specific table bits The sequence provides the direction of research and further studies the structure and function of HA and NA, immunological recognition, construction of HA and NA mutants, and selection of new HA and NA molecules, R & D diagnostic and gene engineering The PCR amplified codon-optimized 16 HA1 and 9 NA genes were then cloned into the reconstructed gp67-pFastBac1 vector, Twenty-five recombinant baculovirus were obtained. The recombinant baculovirus was infected with Sf9 cells respectively, and the supernatant and cells were respectively charged with anti-6-deoxyHis antibody. The results showed that the molecular mass of the expression products of 16 HA1 and 9 NA genes was 52. 5kD and 71. 6kD, respectively. The purity of H5-HA1 and H9-HA1 after Ni-column purification is about 90%, and the recovery rate is divided. in addition, 16 subtypes of HA and 9 subtypes of NA gene were cloned into a 5-type recombinant adenovirus vector, Twenty-five recombinant adenoviruses were obtained. Twenty-five recombinant adenoviruses were immunized against BALB/ c mice by nasal drops. The antibody of the corresponding subtype was prepared. The positive rate was 100% after three immunization, and the antibody effect was detected by ELISA. The target protein of each subtype expressed by baculovirus system was used as antigen, and the serum from BALB/ c mice immunized with recombinant adenovirus was used as an antibody, and 16 HA subtypes and 9 NA subtypes were determined by ELISA. The results showed that the cross reaction between subtypes of HAl and NA was complicated, the cross reaction between H2, H5, H7 subtypes and other subtypes was weak, while H6, H12, H16 were similar to those of other subtypes. Cross-reactivity between subtypes of subtype N1, N2 and other subtypes is strong, and the cross-reactivity between HA1 and NA subtypes is separated from its sequence. there is a certain difference in the result of the analysis. An optimized RT-PCR method was used to amplify the 8 gene fragments of influenza A/ PR/ 8/ 34 (H1N1) strain and cloned into the pIVVII vector. Three silent mutation tags were introduced into the PB1 fragment. Eight functional plasmids were mixed with 293T and MDCK to culture the cells, and the cell lesion, RT-PCR and electron microscopy were used. Experiments prove that the system can successfully save the influenza A virus. In order to improve the rescue efficiency of the influenza A virus, the PIVVS plasmid is constructed by inserting the artificially synthesized SCPI promoter[1] into a PIVVII vector, and the eight gene fragments of A/ PR/ 8/ 34 (H1N1) are cloned into a pIVVS vector, The supernatant and cells of the transfected cells were harvested at 24h, 48h, 72h and 96h post-harvest, respectively. The results showed that the expression of EGFP, NP expression and hemagglutination titer in different time points of the optimized system were higher than those of the unoptimized system.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373
本文编号：2304919
[Abstract]:Influenza A virus is a serious threat to human health, which poses a serious threat to human beings due to the pandemic of influenza A virus. In recent years, people are infected with avian influenza virus (H5N1) and influenza A (H1N1) influenza, and further suggests the importance of strengthening the research and control of influenza A virus. Hemagglutinin (HA) and neuraminase (NA) are the glycoprotein encoded by influenza A virus, which can be further divided into 16 HA subtypes (H1-H16) and 9 NA subtypes according to different antigenicity (N1-N 9). Although HA and NA are the main immunogens of influenza virus, because of the strong variability of HA and NA, the subtypes are numerous, and the immune cross-relationship between the 16 HA and 9 NA subtypes of influenza A virus has not yet been completed. The establishment of specific immune detection technology for influenza virus subtypes and the research and development of vaccine have been put forward. In this study, the cross reactivity of HAl and 9 subtypes of influenza A virus was studied in order to elucidate its immune response characteristics, and to provide a feasible theory for the development of influenza A virus detection technology and universal vaccine. According to the invention, in recent years, the invention of the reverse genetic system provides a key technology platform for artificially modifying the influenza A virus and the R & D vaccine, but the rescue efficiency of the existing system needs to be This study is intended to improve its rescue by modifying the transcription/ expression vector of influenza A virus reverse genetic system. Efficiency. First, Influenza A HA and NA Serological Cross Response Characteristics: A Study of the HA Gene of 16 Subtypes of Influenza A Virus and the NA Gene of 9 Subtypes of Influenza A Virus The results showed that the homology of 16 HA and 9 NA sequences with amino acid sequence was mainly two trend evolutions, and the homology among 16 HA sequences was 28%. Between-76.7%, the homology between the amino acid sequences of the 9 NA subtypes ranged from 43.1% to 70.4%, and the homology between the amino acid sequences was 37. 9. While 16 HA and 9 NA genes predict B cell linear table bit distribution, there are differences in specific table bits, which are serologic cross reactions between subtypes and determine subtype specific table bits The sequence provides the direction of research and further studies the structure and function of HA and NA, immunological recognition, construction of HA and NA mutants, and selection of new HA and NA molecules, R & D diagnostic and gene engineering The PCR amplified codon-optimized 16 HA1 and 9 NA genes were then cloned into the reconstructed gp67-pFastBac1 vector, Twenty-five recombinant baculovirus were obtained. The recombinant baculovirus was infected with Sf9 cells respectively, and the supernatant and cells were respectively charged with anti-6-deoxyHis antibody. The results showed that the molecular mass of the expression products of 16 HA1 and 9 NA genes was 52. 5kD and 71. 6kD, respectively. The purity of H5-HA1 and H9-HA1 after Ni-column purification is about 90%, and the recovery rate is divided. in addition, 16 subtypes of HA and 9 subtypes of NA gene were cloned into a 5-type recombinant adenovirus vector, Twenty-five recombinant adenoviruses were obtained. Twenty-five recombinant adenoviruses were immunized against BALB/ c mice by nasal drops. The antibody of the corresponding subtype was prepared. The positive rate was 100% after three immunization, and the antibody effect was detected by ELISA. The target protein of each subtype expressed by baculovirus system was used as antigen, and the serum from BALB/ c mice immunized with recombinant adenovirus was used as an antibody, and 16 HA subtypes and 9 NA subtypes were determined by ELISA. The results showed that the cross reaction between subtypes of HAl and NA was complicated, the cross reaction between H2, H5, H7 subtypes and other subtypes was weak, while H6, H12, H16 were similar to those of other subtypes. Cross-reactivity between subtypes of subtype N1, N2 and other subtypes is strong, and the cross-reactivity between HA1 and NA subtypes is separated from its sequence. there is a certain difference in the result of the analysis. An optimized RT-PCR method was used to amplify the 8 gene fragments of influenza A/ PR/ 8/ 34 (H1N1) strain and cloned into the pIVVII vector. Three silent mutation tags were introduced into the PB1 fragment. Eight functional plasmids were mixed with 293T and MDCK to culture the cells, and the cell lesion, RT-PCR and electron microscopy were used. Experiments prove that the system can successfully save the influenza A virus. In order to improve the rescue efficiency of the influenza A virus, the PIVVS plasmid is constructed by inserting the artificially synthesized SCPI promoter[1] into a PIVVII vector, and the eight gene fragments of A/ PR/ 8/ 34 (H1N1) are cloned into a pIVVS vector, The supernatant and cells of the transfected cells were harvested at 24h, 48h, 72h and 96h post-harvest, respectively. The results showed that the expression of EGFP, NP expression and hemagglutination titer in different time points of the optimized system were higher than those of the unoptimized system.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R373
【共引文献】
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