【作者】 彭广能；

【导师】 汪开毓； 夏咸柱；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2010， 博士

【摘要】 [目的]构建能够分别表达虎源H5N1亚型流感病毒血凝素(HA)基因和核蛋白(NP)基因的真核表达质粒以及表达HA和NP蛋白重组犬2型腺病毒(CAV-2)活载体疫苗(CAV-2-HA和CAV-2-NP),用所构建的真核表达质粒和活载体疫苗免疫小鼠,检测各质粒和活载体疫苗的免疫水平及其攻毒保护率,通过本实验为哺乳动物H5N1亚型流感病毒核酸疫苗和重组活载体疫苗的应用研究提供理论与技术基础。[方法]利用设计合成的HA和NP引物,从A/Tiger/Harbin/132/2007 (H5N1)毒株的鸡胚尿囊增殖液中对该毒株的HA和NP基因进行PCR扩增、克隆和测序分析；应用常规分子生物学方法,构建能够分别表达H5N1亚型流感病毒HA和NP基因的真核表达质粒pVAX-HA和pVAX-NP以及同时表达猫IL-18基因和H5N1亚型流感病毒NP/HA基因的双表达质粒pIRES2-IL-18-HA和pIRES2-IL-18-NP,应用间接免疫荧光和间接ELISA方法检测各真核表达质粒在乳仓鼠肾传代细胞(BHK-21)中的表达情况。然后将含有NP/HA基因的表达盒(CMV+NP/HA+PolyA)克隆入pVAXE3的SspⅠ酶切缺失处,分别获得含有NP/HA表达盒的穿梭载体pVAXΔE3-NP/HA。用SalⅠ+NruⅠ分别对pVAXΔE3-NP/HA和pPoly2-CAV2进行双酶切,将含有NP/HA表达盒的片段定向克隆入pPoly2-CAV2,获得在E3区缺失处插入NP/HA表达盒的重组质粒pCAV-2-NP/HA。释放CAV-2-NP/HA重组基因组,脂质体介导转染犬肾细胞(DK),获得了能使DK细胞产生典型腺病毒样细胞病变的重组病毒(CAV-2-NP/HA)。从形态学、基因组水平、NP/HA基因的转录、NP/HA蛋白的表达以及重组病毒的生长特征等方面进行鉴定。用所构建的4种真核表达质粒和2种重组病毒分别进行小鼠免疫实验和攻毒保护实验。真核表达质粒的免疫实验共设pVAX-HA、pVAX-NP、pVAX-HA和pVAX-NP联合、pIRES2-IL-18-HA、pIRES2-IL-18-NP、pIRES2-IL-18-HA和pIRES2-IL-18-NP联合、PBS对照7个免疫组,每组11只小鼠,采用肌肉注射的方法,以15天的时间间隔免疫3次后15天,用间接ELISA方法检测各免疫组小鼠产生的抗AIV的抗体水平；用MTT法检测各免疫组小鼠产生的细胞免疫水平；用H5N1亚型流感病毒强毒株滴鼻攻毒,检测各免疫组的攻毒保护率。重组病毒的免疫实验共设CAV-2-HA、CAV-2-HA和CAV-2-NP联合免疫、CAV-2三组,每组13只小鼠。采用滴鼻(50μL)+皮下注射(250μL)的免疫方法,以14天的时间间隔共免疫接种2次。每次免疫后14天,每组各扑杀2只采血收集血清,剩下9只中3只用于攻毒后4天处死,测定攻毒后肺滴度,剩余6只用于检测攻毒后的保护率。[结果]成功克隆了A/Tiger/Harbin/132/2007 (H5N1)毒株的HA和NP全基因,长度分别为1704bp和1479bp,分别编码568和493个氨基酸。成功构建了表达虎源H5N1亚型流感病毒HA和NP蛋白的4种真核表达质粒(pVAX-HA、pVAX-NP、pIRES2-IL-18-HA和pIRES2-IL-18-NP)和2种重组病毒(CAV-2-HA和CAV-2-NP)。这4种真核表达质粒均能在BHK-21细胞中表达具有生物活性的HA和NP蛋白,均能诱导小鼠产生特异性的细胞免疫和体液免疫,各免疫组小鼠在受到超大剂量(10000 MLD50/只)的强毒攻击后,各组免疫保护率分别为：pVAX-HA组60%、pVAX-NP组20%、pVAX-HA和pVAX-NP联合组60%、pIRES2-IL-18-HA组20%、pIRES2-IL-18-NP组0%、pIRES2-IL-18-HA和pIRES2-IL-18-NP联合组20%、PBS对照组0%。CAV-2-NP/HA具有典型的CAV-2形态特征,CAV-2-NP/HA在繁殖的过程中没有对H5N1 NP/HA表达盒片段进行缺失或重排,并且能够转录H5N1 NP/HA的mRNA, ELISA和免疫荧光分析表明重组表达产物可被流感病毒NP/HA单克隆抗体所识别,在体外具有良好的遗传稳定性。动物免疫实验表明CAV-2-HA、CAV-2-NP两种重组活载体疫苗均能诱导小鼠产生特异性的免疫反应,以CAV-2-HA效果最好,对小鼠的攻毒保护率为83.3%,CAV-2-HA和CAV-2-NP联合免疫的保护率为16.7%,对照组(CAV-2)为0%。[结论]本实验首次成功克隆了A/Tiger/Harbin/132/2007毒株的HA和NP全基因；首次成功构建了表达虎源H5N1亚型流感病毒HA和NP蛋白的4种核酸疫苗和2种重组犬2-型腺病毒活载体疫苗,它们均能诱导小鼠产生免疫保护作用,这为哺乳动物H5N1亚型流感病毒核酸疫苗和重组活载体疫苗的应用研究奠定了理论与技术基础。更多还原

【Abstract】 [Objective] Construct the eukaryotic expressing plasmids which can express the hemagglutinin (HA) and neucleoprotein (NP) gene of H5N1 influenza virus and the live trager vaccines(CAV-2-HA and CAV-2-HP) of expressing the HA and NP gene recombinant Canine Adenovirus Type 2. Then use the eukaryotic expressing plasmid and the live trager vaccines to immunize the mice, the humoral immunity and cell-mediated immunity response of the immunized mice are detected and the protective rates of the eukaryotic expressing plasmid is analyzed. The work done here provides a valuable theoretical and technical bases for the nucleic acid vaccine and the recombinant live vector vaccine development against mammalian H5N1 subtype influenza virus.[Method] HA and NP gene of A/Tiger/Harbin/132/2007 (H5N1) from the chick embryo allantoic fluid were amplificated through PCR by the contrivable synthetic primer of HA and NP gene,then cloned and sequencing analyzed. Ordinary molecular methods were used to construct eukaryotic expressing vectors, pVAX-HA and pVAX-NP expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger. Dual-expressing vector pIRES2-IL-18-HA expressing HA gene of H5N1 subtype influenza virus and IL-18 gene of feline was also constructed. The protein expressed by eukaryotic vectors in BHK-21 cell was detected by indirect immunofluorescence assay and indirect ELISA.Then the expression cassette containing NP/HA gene (CMV+NP/HA+PolyA) was cloned into the Ssp I enzyme losses division of pVAXE3,then obtaining shuttle vector (pVAXΔE3-NP/HA) containing NP expression cassette. Using Sal I+Nru I double digestion for pVAXΔE3-NP/HA and pPoly-2-CAV2. The fragment containing NP/HA expression cassette was directionally cloned into pPoly2-CAV2 and obtained recombinant plasmid (pCAV-2-NP/HA) of NP/HA expression cassette inserted into the E3 lacking area. After Releasing CAV-2-NP/HA recombination genome and transfecting DK cells by liposomes mediate, the CAV-2-NP/HA recombination virus which typically made the DK cells adenovirus-like cytopathic. From morphology, genomics level, NP/HA genes transcription, NP/HA protein expression and the growth characteristics of recombinant virus, etc, we indentified the recombinant virus.The mice immunization experiment and conteracting toxic substances and protection experiment were taken with four eukaryotic expressing plasmid and two recombinant viruses respectively. Design steps of eukaryotic expressing plasmid immunization experiment as follow.7 immune groups were used:immunized with pVAX-HA, pVAX-NP, pVAX-Ha and pVAX-NP, pIRES2-IL-18-HA, IRES2-IL-18-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, PBS, respectively.,11 mice in each group. Mice were immunized 3 times with a interval of 15 days by intramuscular injection. 15 days after inoculation the antibody titers of the mice in each group against AIV were detected by indirect ELISA, MTT method was used to detect the cell-mediated immunity response. The mice in each group were inoculated with highly pathogenic H5N1 subtype influenza virus, in order to measure the protective rates.Design steps of the immunization experiment of recombinant virus as follow.3 immune groups were used: immunized with CAV-2-HA、CAV-2, combined immunized with CAV-2-HA and CAV-2-NP. There were 13 mice in each group. Mice were immunized 2 times with a interval of 14 days by drip into the nose (50μL) and subcutaneous injection(250μL).14 days after inoculation of each time, two mice were killed in each group to collect serum and took conteracting toxic substances in 3 mice of each group to detect lung titer four days later. The remaining was used to detect the protective rate after conteracting toxic substances.[Result] The total NP and HA gene of A/Tiger/Harbin/132/2007 strain are cloned successfully, the lengths are 1704bp and 1479bp, each of them encodes 568 and 493 amine acids respectively. Four eukaryotic expressing plasmids (pVAX-HA, pVAX-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP)of expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger and two recombinant viruses(CAV-2-NP and CAV-2-HA) are constructed successfully. These four eukaryotic expressing vectors all have the ability to express HA and NP protein with biological activity in the mammalian cell, BHK-21. Mice in each immunized group are inoculated high titer of highly pathogenic virus (10000 MLD50/each), the protective rates of pVAX-HA, pVAX-NP, pVAX-HA and pVAX-NP, pIRES2-IL-18-HA, pIRES2-IL-18-NP, pIRES2-IL-18-HA and pIRES2-IL-18-NP, PBS immunized group are 60%,20%,60%,20%,0%,20%,0%, respectively.CAV-2-NP/HA has the typical morphological characteristics of CAV-2, the fragment of H5N1 NP/HA expression cassette is not missing or resort in the process of reproduction of CAV-2-NP/HA, and can transcribes the mRNA of H5N1 NP/HA, ELISA and immunofluorescence analysis show that the product of recombinant expression can be identified by the NP/HA monoclonal antibody of flu virus, has a good in vitro genetic stability. The animal immunization experiment shows that the two recombinant vaccines can induce mice to create specific immunereaction and CAV-2-HA is better than the other one, its conteracting toxic substances protective rate to mice is 83.3%, the protective rate of combined immunization of CAV-2-HA and CAV-2-NP is 16.7%, control group(CAV-2) is 0%.[Conclusion] The total NP and HA gene of A/Tiger/Harbin/132/2007 strain is cloned successfully; Four nucleic acid vaccines and two recombinant live vector vaccines of expressing HA and NP gene of H5N1 subtype influenza virus isolated from tiger are constructed successfully, they all can introduce mice to create the immunoprotection effect. This research provides a valuable theoretical and technical bases for the nucleic acid vaccine and recombinant live vector vaccines development against mammalian H5N1 subtype influenza virus.更多还原