基于蛋白轉(zhuǎn)導(dǎo)域的人禽流感復(fù)制子疫苗的構(gòu)建和誘導(dǎo)細(xì)胞免疫應(yīng)答研究
發(fā)布時(shí)間：2019-06-14 22:43
【摘要】： 目的 本課題通過(guò)生物信息學(xué)手段比較分析A/Anhui/1/2005毒株HA基因抗原的免疫原性和代表性,并整合復(fù)制子具有自我復(fù)制和高效表達(dá)外源抗原特點(diǎn)以及蛋白轉(zhuǎn)導(dǎo)域VP22蛋白的轉(zhuǎn)導(dǎo)功能的優(yōu)勢(shì),構(gòu)建和包裝人禽流感復(fù)制子疫苗,以期增強(qiáng)禽流感復(fù)制子疫苗誘導(dǎo)的免疫應(yīng)答,尤其是抗原特異性CD8~+ T淋巴細(xì)胞介導(dǎo)的特異性CTL反應(yīng),為研制高效、安全及通用性禽流感人疫苗臨床應(yīng)用奠定實(shí)驗(yàn)基礎(chǔ)。 材料與方法 首先,檢索1997 2008年流感病毒數(shù)據(jù)庫(kù)(influenza virus resources)收錄全部甲型流感病毒(H5N1亞型)人源分離株的HA基因序列和氨基酸序列。采用系統(tǒng)進(jìn)化樹(shù)分析A/Anhui/1/2005與其它各毒株之間親緣關(guān)系。采用DNASTAR、DNAMAN軟件及相關(guān)在線分析蛋白抗原指數(shù)和抗原表位。 其次,擴(kuò)增禽流感病毒(H5N1)人源分離株的HA基因及VP22和EGFP基因的全長(zhǎng)編碼序列,同時(shí)采用剪切重疊延伸PCR(SOE-PCR,splicing by overlapextension-PCR)技術(shù)擴(kuò)增VP22和HA的融合基因(VP22/HA),將HA、VP22、EGFP基因及VP22/HA融合基因分別構(gòu)建到pSFV載體26S亞基因組下游,構(gòu)建pSFV-HA、pSFV-VP22、pSFV-EGFP和pSFV-VP22/HA質(zhì)粒。將構(gòu)建的質(zhì)粒進(jìn)行體外轉(zhuǎn)錄,并在BHK-21細(xì)胞中包裝成VRP-HA、VRP-VP22、VRP-EGFP、VRP-VP22/HA復(fù)制子。將包裝的各復(fù)制子感染BHK-21細(xì)胞后,在熒光顯微鏡下觀察EGFP表達(dá)情況,采用RT-PCR和間接免疫熒光染色法(IFA)檢測(cè)HA、VP22及VP22/HA蛋白表達(dá)情況。同時(shí),采用Annexin V/PI雙染色法檢測(cè)細(xì)胞凋亡情況。 最后,分別用不同劑量的VRP-HA及VRP-VP22/HA復(fù)制子疫苗及相應(yīng)的VRP-VP22復(fù)制子、VRP-EGFP復(fù)制子和生理鹽水對(duì)照免疫6-8周齡BALB/c雌性小鼠,經(jīng)右側(cè)大腿肌肉注射初次免疫和3周后加強(qiáng)免疫,加強(qiáng)免疫2周后在乙醚麻醉下處死小鼠分離脾臟,分離淋巴細(xì)胞。采用流式細(xì)胞儀檢測(cè)CD4~+T細(xì)胞表達(dá)IL-4和CD8~+ T細(xì)胞表達(dá)IFN-γ等細(xì)胞內(nèi)細(xì)胞因子表達(dá)情況。采用SPSS 13.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,數(shù)據(jù)以均數(shù)(M)±標(biāo)準(zhǔn)差(SD),表達(dá)細(xì)胞因子的細(xì)胞百分比分析采用近似正態(tài)法單因素方差分析(ANOVA),多組兩兩比較采用SNK法。 結(jié)果 對(duì)1997-2008年的232株參考序列進(jìn)行系統(tǒng)發(fā)生樹(shù)分析發(fā)現(xiàn)A/Anhui/1/2005候選株的HA基因與國(guó)內(nèi)及印尼等國(guó)的人禽流感病毒分離株親緣關(guān)系最近;該候選株與泰國(guó)和越南等國(guó)的人禽流感病毒分離株的親緣關(guān)系也相對(duì)較近。對(duì)各亞系相對(duì)構(gòu)成分析發(fā)現(xiàn),A/Anhui/1/2005候選株所屬亞系中同系分離株數(shù)量占總分離株數(shù)量的70.0%(162/232,當(dāng)遺傳距離為0.0245時(shí)),當(dāng)包含泰國(guó)和越南等國(guó)的人禽流感病毒分離株時(shí)(遺傳距離為0.0175),其所屬亞系中同源分離株數(shù)量占總分離株數(shù)量的91.8%(213/232)。對(duì)國(guó)內(nèi)14株H5N1禽流感病毒人源分離株HA基因的核苷酸和氨基酸的同源性分析,發(fā)現(xiàn)其核苷酸和氨基酸的同源性分別在94.7%-100%和95.3%-100%之間�？乖砦活A(yù)測(cè)表明,疫苗候選株A/Anhui/1/2005(ABD28180)有26個(gè)抗原表位,為國(guó)內(nèi)分離株抗原表位最多毒株之一,抗原表位多數(shù)都處在親水區(qū)段,且較少處在糖基化位點(diǎn)區(qū)段。 將構(gòu)建的表達(dá)目的基因的復(fù)制子載體進(jìn)行測(cè)序,顯示pSFV復(fù)制子載體中HA、VP22、EGFP基因及VP22/HA融合基因序列與理論序列完全一致,并位于pSFV載體的亞基因啟動(dòng)子的下游。經(jīng)瓊脂糖變性膠電泳發(fā)現(xiàn)pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA和pSFV helper載體的轉(zhuǎn)錄產(chǎn)物分別約為9500 bp,8700 bp,8500 bp,10400 bp and 7000左右,并且沒(méi)有明顯拖尾現(xiàn)象。經(jīng)直接熒光顯微鏡、RT-PCR和間接免疫熒光檢測(cè)發(fā)現(xiàn),各復(fù)制子感染的細(xì)胞均表達(dá)其相應(yīng)目的基因;而且,經(jīng)間接免疫熒光檢測(cè)結(jié)果發(fā)現(xiàn),VRP-VP22/HA感染的BHK-21細(xì)胞比VRP-HA復(fù)制子感染的BHK-21細(xì)胞產(chǎn)生更強(qiáng)、更密集的熒光強(qiáng)度。凋亡檢測(cè)發(fā)現(xiàn),各復(fù)制子感染的BHK-21細(xì)胞均出現(xiàn)明顯凋亡現(xiàn)象,尤其是早期凋亡。 VRP-HA和VRP-VP22/HA兩種復(fù)制子疫苗經(jīng)10~5TU和10~6TU兩種劑量的免疫6-8周齡BALB/c雌性小鼠后,發(fā)現(xiàn)VRP-HA和VRP-VP22/HA復(fù)制子疫苗免疫的小鼠脾臟CD4~+T細(xì)胞高水平表達(dá)IL-4細(xì)胞因子,CD8~+T細(xì)胞高水平表達(dá)IFN-γ細(xì)胞因子,而對(duì)照組(VRP-VP22,VRP-EGFP和NS)中兩種細(xì)胞因子呈現(xiàn)低水平表達(dá);其中,10~6-VRP-HA,10~5-和10~6-VRP-VP22/HA免疫組是顯著高于VRP-VP22,VRP-EGFP和NS對(duì)照組,差異有顯著性(p0.01)。而10~5TU的VRP-HA疫苗免疫組誘導(dǎo)表達(dá)IL-4和IFN-γ與對(duì)照組差異無(wú)顯著性。而且,10~6-VRP-VP22/HA復(fù)制子疫苗免疫組高于10~5-VRP-VP22/HA復(fù)制子疫苗組,后者又高于10~6-VRP-HA復(fù)制子疫苗組。 結(jié)論 采用A/Anhui/1/2005毒株的HA基因構(gòu)建疫苗具有普遍的代表性,抗原性預(yù)測(cè)表明該毒株HA基因具有較高抗原性。 pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA質(zhì)粒均已正確構(gòu)建,并成功包裝了VRP-HA、VRP-VP22、VRP-EGFP和VRP-VP22/HA復(fù)制子,各復(fù)制子均可誘導(dǎo)細(xì)胞凋亡。 VRP-HA和VRP-VP22/HA復(fù)制子疫苗均能誘導(dǎo)IL-4、IFN-γ細(xì)胞因子表達(dá),并呈現(xiàn)一種劑量效應(yīng)關(guān)系;VP22基因與HA基因融合的復(fù)制子疫苗優(yōu)于單HA基因的復(fù)制子疫苗。
[Abstract]:Purpose In this paper, the immunogenicity and representativeness of the HA gene antigen of A/ Anhua/1/2005 strain are compared and analyzed by means of bioinformatics, and the whole replicon has the characteristics of self-replication and high-efficiency expression of the foreign antigen and the transduction function of the protein transduction domain VP22 protein. The invention has the advantages that the human avian influenza replicon vaccine is constructed and packaged with the aim of enhancing the immune response induced by the avian influenza replicon vaccine, in particular the specific CTL reaction mediated by the antigen-specific CD8 + T lymphocyte, and laying an experiment for the development of a high-efficiency, safe and general-purpose avian influenza vaccine clinical application. The foundation. Materials and methods First, the HA gene of all human isolates of influenza A virus (H5N1 subtype) was retrieved from the influenza virus database in 2008. Sequence and amino acid sequence. A/ Anhua/1/2005 and others are analyzed using the phylogenetic tree The genetic relationship among the strains was determined by the DNA STAR, DNMAN software and the related on-line analysis protein. Next, the full length coding sequence of the HA gene and the VP22 and the EGFP gene of the human isolated strain of the avian influenza virus (H5N1) is amplified, and the fusion gene (VP22/ H) of the VP22 and the HA is amplified by using a shear overlap extension PCR (SOE-PCR) technology. A) constructing pSFV-HA, pSFV-VP22, pSFV-EGFP and pSF by respectively constructing the HA, VP22, EGFP gene and VP22/ HA fusion gene to the downstream of the pSFV vector 26S sub-genome; V-VP22/ HA plasmid. The constructed plasmid was transcribed in vitro and packaged into VRP-HA, VRP-VP22, VRP-EGFP, VRP in BHK-21 cells. -VP22/ HA replicon. After each replicon of the package was infected with BHK-21 cells, EGFP expression was observed under a fluorescence microscope, and HA, VP22, and VP were detected by RT-PCR and indirect immunofluorescence staining (IFA). 22/ HA protein expression, while Annexin V/ PI double The apoptosis of the cells was detected by the staining method. The VRP-HA and the VRP-VP22/ HA replicon vaccine and the corresponding VRP-VP22 replicon, the VRP-EGFP replicon and the normal saline control were used to immunize 6-8-week-old BALB/ c female mice. After the first and third weeks of the injection, the immunization was enhanced, and after 2 weeks of booster immunization, the patient was under the anesthesia of ether. The expression of IL-4 and CD8 ~ + T cells in CD4 ~ + T cells was detected by flow cytometry. The data were analyzed by SPSS 13.0, and the mean number (M) and standard deviation (SD) and the percentage of the cells expressing the cytokines were analyzed by means of the approximate normal method and one-factor analysis of variance (ANOVA). ), The results showed that the HA gene of A/ Anhui/1/2005 candidate strain was compared with that of China and China by using the SNK method. The relationship between the human avian influenza virus isolates in Indonesia and other countries is most recent, and the candidate strain is similar to that of Thailand and Vietnam The relationship between the isolates of avian influenza virus was relatively close to each other. The relative composition of each subline found that the number of co-system isolates in the A/ Anhua/1/2005 candidate strain accounted for 70.0% of the total isolates (162/232, when the genetic distance was 0.0245), when the human avian influenza in countries such as Thailand and Vietnam were included When the isolated strain of the virus was isolated (the genetic distance was 0.0175), the number of homologous isolates in the subseries was the total. 91.8% (213/232) of the number of isolates (213/232). The homology of the nucleotide and amino acids of the HA gene of the human isolated strain of the 14 strains of the H5N1 avian influenza virus in China was analyzed, and the homology of the nucleotide and the amino acid of the HA gene was found to be 94. The vaccine candidate A/ Anhua/1/2005 (ABD28180) has 26 antigen epitopes, one of the most virulent strains of the domestic isolates and the majority of the epitope. And sequencing the replicon vector of the constructed expression target gene to display the sequence of HA, VP22, EGFP gene and VP22/ HA fusion gene in the pSFV replicon vector and the theoretical sequence. The transcription products of pSFV-HA, pSFV-VP22, pSFV-EGFP, pSFV-VP22-HA and pSFV helper vector were found to be about 9500 bp,8700 bp,8500 bp,1040, respectively. The results of direct fluorescence microscopy, RT-PCR and indirect immunofluorescence showed that the infected cells of each replicon express their corresponding target genes; moreover, the results of indirect immunofluorescence test showed that the BHK-21 cell ratio of VRP-VP22/ HA was higher than that of V. RP-HA replicon sense The stained BHK-21 cells produced a stronger, more intense fluorescence intensity. The apoptosis test found that the BHs of the replicon infections After 6-8-week-old BALB/ c female mice with two doses of VRP-HA and VRP-VP22/ HA, both VRP-HA and VRP-VP22/ HA replicon vaccines were immunized with two doses of 10-5TU and 10-6TU for 6-8-week-old BALB/ c female mice, and the high-level expression of IL-4, CD8 + T cells and high water was found in the spleen CD4 ~ + T cells of mice immunized with VRP-HA and VRP-VP22/ HA replicon vaccine. The levels of IL-6-VRP-HA,10-5-and 10-6-VRP-VP22/ HA were significantly higher than that of VRP-VP22, VR in the control group (VRP-VP22, VRP-EGFP and NS). P-EGFP and NS control group, the difference was significant (p0.01), while the VRP-HA vaccine of 10-5TU was free of use. There was no significant difference in the expression of IL-4 and IFN-2 in the epidemic group, and the 10-6-VRP-VP22/ HA replicon vaccine immunized group was higher than 10-5-VRP-VP22/ HA replicon. VACCINE Group, the latter was higher than that of the 10-6-VRP-HA replicon vaccine group. Conclusion The HA gene of A/ Anhua/1/2005 strain was constructed. The VRP-HA, pSFV-VP22, pSFV-EGFP and pSFV-VP22-HA plasmids were constructed correctly and the VRP-HA, VRP-VP22, V were successfully packaged. Both the RP-EGFP and the VRP-VP22/ HA replicon, each of which can induce apoptosis. The VRP-HA and the VRP-VP22/ HA replicon vaccines are capable of inducing IL-4, IFN-derived cytokine expression, and presenting an agent
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
本文編號(hào)：2499755
[Abstract]:Purpose In this paper, the immunogenicity and representativeness of the HA gene antigen of A/ Anhua/1/2005 strain are compared and analyzed by means of bioinformatics, and the whole replicon has the characteristics of self-replication and high-efficiency expression of the foreign antigen and the transduction function of the protein transduction domain VP22 protein. The invention has the advantages that the human avian influenza replicon vaccine is constructed and packaged with the aim of enhancing the immune response induced by the avian influenza replicon vaccine, in particular the specific CTL reaction mediated by the antigen-specific CD8 + T lymphocyte, and laying an experiment for the development of a high-efficiency, safe and general-purpose avian influenza vaccine clinical application. The foundation. Materials and methods First, the HA gene of all human isolates of influenza A virus (H5N1 subtype) was retrieved from the influenza virus database in 2008. Sequence and amino acid sequence. A/ Anhua/1/2005 and others are analyzed using the phylogenetic tree The genetic relationship among the strains was determined by the DNA STAR, DNMAN software and the related on-line analysis protein. Next, the full length coding sequence of the HA gene and the VP22 and the EGFP gene of the human isolated strain of the avian influenza virus (H5N1) is amplified, and the fusion gene (VP22/ H) of the VP22 and the HA is amplified by using a shear overlap extension PCR (SOE-PCR) technology. A) constructing pSFV-HA, pSFV-VP22, pSFV-EGFP and pSF by respectively constructing the HA, VP22, EGFP gene and VP22/ HA fusion gene to the downstream of the pSFV vector 26S sub-genome; V-VP22/ HA plasmid. The constructed plasmid was transcribed in vitro and packaged into VRP-HA, VRP-VP22, VRP-EGFP, VRP in BHK-21 cells. -VP22/ HA replicon. After each replicon of the package was infected with BHK-21 cells, EGFP expression was observed under a fluorescence microscope, and HA, VP22, and VP were detected by RT-PCR and indirect immunofluorescence staining (IFA). 22/ HA protein expression, while Annexin V/ PI double The apoptosis of the cells was detected by the staining method. The VRP-HA and the VRP-VP22/ HA replicon vaccine and the corresponding VRP-VP22 replicon, the VRP-EGFP replicon and the normal saline control were used to immunize 6-8-week-old BALB/ c female mice. After the first and third weeks of the injection, the immunization was enhanced, and after 2 weeks of booster immunization, the patient was under the anesthesia of ether. The expression of IL-4 and CD8 ~ + T cells in CD4 ~ + T cells was detected by flow cytometry. The data were analyzed by SPSS 13.0, and the mean number (M) and standard deviation (SD) and the percentage of the cells expressing the cytokines were analyzed by means of the approximate normal method and one-factor analysis of variance (ANOVA). ), The results showed that the HA gene of A/ Anhui/1/2005 candidate strain was compared with that of China and China by using the SNK method. The relationship between the human avian influenza virus isolates in Indonesia and other countries is most recent, and the candidate strain is similar to that of Thailand and Vietnam The relationship between the isolates of avian influenza virus was relatively close to each other. The relative composition of each subline found that the number of co-system isolates in the A/ Anhua/1/2005 candidate strain accounted for 70.0% of the total isolates (162/232, when the genetic distance was 0.0245), when the human avian influenza in countries such as Thailand and Vietnam were included When the isolated strain of the virus was isolated (the genetic distance was 0.0175), the number of homologous isolates in the subseries was the total. 91.8% (213/232) of the number of isolates (213/232). The homology of the nucleotide and amino acids of the HA gene of the human isolated strain of the 14 strains of the H5N1 avian influenza virus in China was analyzed, and the homology of the nucleotide and the amino acid of the HA gene was found to be 94. The vaccine candidate A/ Anhua/1/2005 (ABD28180) has 26 antigen epitopes, one of the most virulent strains of the domestic isolates and the majority of the epitope. And sequencing the replicon vector of the constructed expression target gene to display the sequence of HA, VP22, EGFP gene and VP22/ HA fusion gene in the pSFV replicon vector and the theoretical sequence. The transcription products of pSFV-HA, pSFV-VP22, pSFV-EGFP, pSFV-VP22-HA and pSFV helper vector were found to be about 9500 bp,8700 bp,8500 bp,1040, respectively. The results of direct fluorescence microscopy, RT-PCR and indirect immunofluorescence showed that the infected cells of each replicon express their corresponding target genes; moreover, the results of indirect immunofluorescence test showed that the BHK-21 cell ratio of VRP-VP22/ HA was higher than that of V. RP-HA replicon sense The stained BHK-21 cells produced a stronger, more intense fluorescence intensity. The apoptosis test found that the BHs of the replicon infections After 6-8-week-old BALB/ c female mice with two doses of VRP-HA and VRP-VP22/ HA, both VRP-HA and VRP-VP22/ HA replicon vaccines were immunized with two doses of 10-5TU and 10-6TU for 6-8-week-old BALB/ c female mice, and the high-level expression of IL-4, CD8 + T cells and high water was found in the spleen CD4 ~ + T cells of mice immunized with VRP-HA and VRP-VP22/ HA replicon vaccine. The levels of IL-6-VRP-HA,10-5-and 10-6-VRP-VP22/ HA were significantly higher than that of VRP-VP22, VR in the control group (VRP-VP22, VRP-EGFP and NS). P-EGFP and NS control group, the difference was significant (p0.01), while the VRP-HA vaccine of 10-5TU was free of use. There was no significant difference in the expression of IL-4 and IFN-2 in the epidemic group, and the 10-6-VRP-VP22/ HA replicon vaccine immunized group was higher than 10-5-VRP-VP22/ HA replicon. VACCINE Group, the latter was higher than that of the 10-6-VRP-HA replicon vaccine group. Conclusion The HA gene of A/ Anhua/1/2005 strain was constructed. The VRP-HA, pSFV-VP22, pSFV-EGFP and pSFV-VP22-HA plasmids were constructed correctly and the VRP-HA, VRP-VP22, V were successfully packaged. Both the RP-EGFP and the VRP-VP22/ HA replicon, each of which can induce apoptosis. The VRP-HA and the VRP-VP22/ HA replicon vaccines are capable of inducing IL-4, IFN-derived cytokine expression, and presenting an agent
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 呂燕波,萬(wàn)瑛,吳玉章;SARS病毒基因組所編碼的E蛋白的二級(jí)結(jié)構(gòu)和B細(xì)胞表位預(yù)測(cè)[J];免疫學(xué)雜志;2003年06期
2 肖明;生物信息學(xué)及其在病毒研究中的應(yīng)用[J];上海師范大學(xué)學(xué)報(bào)(自然科學(xué)版);2003年03期
3 李仁清;魯寧;鄧瑤;王文玲;辛偉;張相民;阮力;;流感病毒RNA聚合酶蛋白PB1在小鼠中可誘導(dǎo)亞型間交叉免疫保護(hù)[J];中華微生物學(xué)和免疫學(xué)雜志;2006年04期
，本文編號(hào)：2499755
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2499755.html
最近更新
教材專著
