基于蛋白轉導域的人禽流感復制子疫苗的構建和誘導細胞免疫應答研究
發(fā)布時間：2019-06-14 22:43
【摘要】： 目的 本課題通過生物信息學手段比較分析A/Anhui/1/2005毒株HA基因抗原的免疫原性和代表性,并整合復制子具有自我復制和高效表達外源抗原特點以及蛋白轉導域VP22蛋白的轉導功能的優(yōu)勢,構建和包裝人禽流感復制子疫苗,以期增強禽流感復制子疫苗誘導的免疫應答,尤其是抗原特異性CD8~+ T淋巴細胞介導的特異性CTL反應,為研制高效、安全及通用性禽流感人疫苗臨床應用奠定實驗基礎。 材料與方法 首先,檢索1997 2008年流感病毒數(shù)據庫(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)進化樹分析A/Anhui/1/2005與其它各毒株之間親緣關系。采用DNASTAR、DNAMAN軟件及相關在線分析蛋白抗原指數(shù)和抗原表位。 其次,擴增禽流感病毒(H5N1)人源分離株的HA基因及VP22和EGFP基因的全長編碼序列,同時采用剪切重疊延伸PCR(SOE-PCR,splicing by overlapextension-PCR)技術擴增VP22和HA的融合基因(VP22/HA),將HA、VP22、EGFP基因及VP22/HA融合基因分別構建到pSFV載體26S亞基因組下游,構建pSFV-HA、pSFV-VP22、pSFV-EGFP和pSFV-VP22/HA質粒。將構建的質粒進行體外轉錄,并在BHK-21細胞中包裝成VRP-HA、VRP-VP22、VRP-EGFP、VRP-VP22/HA復制子。將包裝的各復制子感染BHK-21細胞后,在熒光顯微鏡下觀察EGFP表達情況,采用RT-PCR和間接免疫熒光染色法(IFA)檢測HA、VP22及VP22/HA蛋白表達情況。同時,采用Annexin V/PI雙染色法檢測細胞凋亡情況。 最后,分別用不同劑量的VRP-HA及VRP-VP22/HA復制子疫苗及相應的VRP-VP22復制子、VRP-EGFP復制子和生理鹽水對照免疫6-8周齡BALB/c雌性小鼠,經右側大腿肌肉注射初次免疫和3周后加強免疫,加強免疫2周后在乙醚麻醉下處死小鼠分離脾臟,分離淋巴細胞。采用流式細胞儀檢測CD4~+T細胞表達IL-4和CD8~+ T細胞表達IFN-γ等細胞內細胞因子表達情況。采用SPSS 13.0統(tǒng)計軟件對數(shù)據進行統(tǒng)計分析,數(shù)據以均數(shù)(M)±標準差(SD),表達細胞因子的細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。 結果 對1997-2008年的232株參考序列進行系統(tǒng)發(fā)生樹分析發(fā)現(xiàn)A/Anhui/1/2005候選株的HA基因與國內及印尼等國的人禽流感病毒分離株親緣關系最近;該候選株與泰國和越南等國的人禽流感病毒分離株的親緣關系也相對較近。對各亞系相對構成分析發(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,當遺傳距離為0.0245時),當包含泰國和越南等國的人禽流感病毒分離株時(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)。對國內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測表明,疫苗候選株A/Anhui/1/2005(ABD28180)有26個抗原表位,為國內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區(qū)段。 將構建的表達目的基因的復制子載體進行測序,顯示pSFV復制子載體中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載體的亞基因啟動子的下游。經瓊脂糖變性膠電泳發(fā)現(xiàn)pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA和pSFV helper載體的轉錄產物分別約為9500 bp,8700 bp,8500 bp,10400 bp and 7000左右,并且沒有明顯拖尾現(xiàn)象。經直接熒光顯微鏡、RT-PCR和間接免疫熒光檢測發(fā)現(xiàn),各復制子感染的細胞均表達其相應目的基因;而且,經間接免疫熒光檢測結果發(fā)現(xiàn),VRP-VP22/HA感染的BHK-21細胞比VRP-HA復制子感染的BHK-21細胞產生更強、更密集的熒光強度。凋亡檢測發(fā)現(xiàn),各復制子感染的BHK-21細胞均出現(xiàn)明顯凋亡現(xiàn)象,尤其是早期凋亡。 VRP-HA和VRP-VP22/HA兩種復制子疫苗經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復制子疫苗免疫的小鼠脾臟CD4~+T細胞高水平表達IL-4細胞因子,CD8~+T細胞高水平表達IFN-γ細胞因子,而對照組(VRP-VP22,VRP-EGFP和NS)中兩種細胞因子呈現(xiàn)低水平表達;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對照組,差異有顯著性(p0.01)。而10~5TU的VRP-HA疫苗免疫組誘導表達IL-4和IFN-γ與對照組差異無顯著性。而且,10~6-VRP-VP22/HA復制子疫苗免疫組高于10~5-VRP-VP22/HA復制子疫苗組,后者又高于10~6-VRP-HA復制子疫苗組。 結論 采用A/Anhui/1/2005毒株的HA基因構建疫苗具有普遍的代表性,抗原性預測表明該毒株HA基因具有較高抗原性。 pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA質粒均已正確構建,并成功包裝了VRP-HA、VRP-VP22、VRP-EGFP和VRP-VP22/HA復制子,各復制子均可誘導細胞凋亡。 VRP-HA和VRP-VP22/HA復制子疫苗均能誘導IL-4、IFN-γ細胞因子表達,并呈現(xiàn)一種劑量效應關系;VP22基因與HA基因融合的復制子疫苗優(yōu)于單HA基因的復制子疫苗。
[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
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392
本文編號：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
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392
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