甲型流感病毒亞型間血清學交叉反應特征研究及反向遺傳系統(tǒng)的優(yōu)化
發(fā)布時間：2018-11-01 19:48
【摘要】：甲型流感病毒嚴重危害人類健康,由于其變異引發(fā)的大流行對人類造成嚴重威脅,近年來人感染禽流感病毒(H5N1)和甲型H1N1流感的流行,進一步提示加強甲型流感病毒的研究和控制的重要性。 血凝素(HA)和神經(jīng)氨酸酶(NA)是甲型流感病毒編碼的糖蛋白,變異性強,根據(jù)抗原性不同可進一步分為16個HA亞型(H1-H16)和9個NA亞型(N1-N9)。盡管HA和NA是流感病毒主要的免疫原,但由于HA和NA的變異性強,亞型眾多,而目前甲型流感病毒16個HA和9個NA不同亞型之間的免疫交叉關系尚未系統(tǒng)研究,對流感病毒亞型特異性免疫檢測技術的建立和疫苗的研發(fā)提出了嚴峻的考驗。為此,本研究對甲型流感病毒16個亞型的HAl和9個亞型NA的交叉反應性進行了研究,以期闡明其免疫反應特征,為甲型流感病毒免疫檢測技術和通用疫苗的研制提供可行性的理論依據(jù)。另一方面,近年來反向遺傳系統(tǒng)的發(fā)明為人工改造甲型流感病毒和研發(fā)疫苗提供了關鍵技術平臺,但現(xiàn)有系統(tǒng)的拯救效率有待提高,本研究擬通過對甲型流流感病毒反向遺傳系統(tǒng)的轉(zhuǎn)錄／表達載體的改建提高其拯救效率。 一、甲型流感病毒HA和NA血清學交叉反應特征研究 對本實驗選取的甲型流感病毒16個亞型的HA基因和9個亞型的NA基因的序列進行了分析,結(jié)果表明,16個HA和9個NA的核苷酸與氨基酸序列的進化樹都主要向兩個趨勢進化,16個HA核苷酸序列之間的同源率在28%-76.7%之間,其氨基酸序列之間的同源率在36.2%-78.5%之間,9個NA亞型核苷酸序列之間的同源率在43.1%-70.4%之間,其氨基酸序列之間的同源率在37.9%66.6%之間。盡管16個HA和9個NA基因的預測B細胞線性表位分布區(qū)域一致,但其具體表位組成卻存在差異,這些信息為進行各亞型之間的血清學交叉反應以及確定亞型特異性表位序列提供研究方向,進而為研究HA和NA的結(jié)構(gòu)與功能、免疫識別、構(gòu)建HA和NA的突變體以及選擇表達新型HA和NA分子、研發(fā)診斷和基因工程疫苗奠定了基礎。 隨后將PCR擴增的經(jīng)密碼子優(yōu)化的16個HA1和9個NA基因克隆到改建的gp67-pFastBac1載體中,獲得了25個重組桿狀病毒。將重組桿狀病毒分別感染Sf9細胞,分別收取上清和細胞用抗6×His抗體進行Western blot分析。結(jié)果表明,16個HA1和9個NA基因的表達產(chǎn)物分子質(zhì)量分別約為52.5kD與71.6kD,與預期相符,并且16個HA1和9個NA基因還呈分泌性表達。其中H5-HA1和H9-HA1經(jīng)Ni柱純化后的純度達90%左右,回收率分別為0.5%和0.9%。同時,將16個亞型的HA和9個亞型的NA基因克隆到5型重組腺病毒載體中,得到了25個重組腺病毒。分別將25個重組腺病毒通過滴鼻途徑免疫BALB/c小鼠,制備了相應亞型的抗體,三次免疫后ELISA檢測陽轉(zhuǎn)率為100%,ELISA檢測的抗體效價約為1：6400左右。利用桿狀病毒系統(tǒng)表達的各個亞型的目的蛋白作為抗原,利用重組腺病毒免疫BALB/c小鼠制備的血清作為抗體,通過ELISA方法確定16個HA亞型及9個NA亞型之間的血清學交叉反應關系。結(jié)果表明,HAl和NA各亞型之間的交叉反應關系錯綜復雜,H2、H5、H7亞型與其他亞型之間的交叉反應弱,而H6、H12、H16與其他亞型之間的交叉反應強。N1、N2亞型與其他亞型之間的交叉反應強,HA1和NA各亞型之間的交叉反應結(jié)果與其序列分析的結(jié)果存在一定的差異。 二、甲型流感病毒反向遺傳系統(tǒng)的優(yōu)化 利用RT-PCR的方法擴增了甲型流感病毒A/PR/8/34(H1N1)毒株8個基因片斷,克隆入pIVVII載體中,其中在PB1片段中引入了3個沉默突變標簽。將8個功能質(zhì)粒共轉(zhuǎn)染293T和MDCK混合培養(yǎng)細胞,利用細胞病變、RT-PCR、電鏡技術等證明該系統(tǒng)可成功拯救甲型流感病毒。為提高甲型流感病毒的拯救效率,用人工合成的SCPI啟動子[1]插入PIVVII載體,構(gòu)建出pIVVS質(zhì)粒,再將A/PR/8/34(H1N1)的8個基因片斷克隆入pIVVS載體中,把優(yōu)化后的8個功能質(zhì)粒共轉(zhuǎn)染293T和MDCK混合細胞,在共轉(zhuǎn)染后的24h、48h、72h及96h分別收獲轉(zhuǎn)染細胞的上清和細胞,通過EGFP表達量、NP表達的時相變化以及血凝試驗鑒定兩個包裝系統(tǒng)的拯救效率。結(jié)果表明,優(yōu)化后系統(tǒng)不同時間點的EGFP表達量、NP表達量及血凝效價均高于未優(yōu)化系統(tǒng),說明利用高強度的SCPI啟動子可有效提高甲型流感病毒的包裝效率,為改進甲型流感病毒疫苗的研發(fā)系統(tǒng)奠定了基礎。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R373
本文編號：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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R373
【共引文獻】
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2 林曉燕;人端粒酶逆轉(zhuǎn)錄酶腫瘤疫苗研究[D];中國協(xié)和醫(yī)科大學;2006年
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