HIV复合表位核酸和痘苗病毒载体疫苗构建及免疫原性研究
发布时间：2018-09-11 11:02
【摘要】：鉴于当前获得性免疫缺陷综合征（Acquired immunodeficiency syndrome, AIDS）的流行态势和预防治疗现状，研发安全有效的艾滋病预防/治疗性疫苗刻不容缓。近些年来，很多科学家利用不同的疫苗载体和形式、不同的人免疫缺陷病毒（Human immunodeficiency virus，HIV）免疫原、不同的接种或免疫途径进行了大量的有益的尝试。本研究选择DNA质粒和痘苗病毒天坛株作为免疫原递送系统，开展艾滋病疫苗的免疫原性研究。 本研究在本研究室多年来筛选获得的HIV复合多表位基因（MEGNp24）的基础上，在其上游引入CpG基序和CTB基因作为佐剂，构建了一种重组核酸疫苗pVL-CCMp24，将重组DNA质粒转染293T细胞，从RT-PCR和间接免疫荧光检测结果可知，重组质粒编码的目的基因CCMp24在哺乳动物细胞内得到正确转录和表达。肌肉注射途径免疫BALB/c小鼠，分析重组DNA疫苗的免疫原性，由对HIV特异性抗体、Th1型和Th2型细胞因子、T细胞亚型、淋巴细胞增殖等免疫指标的检测结果表明，重组DNA候选疫苗pVL-CCMp24可以诱导机体产生一定程度的细胞和体液免疫应答。 基于多价疫苗的设计理念，本研究首先构建了一种含三个表达盒的痘苗病毒穿梭载体pSTKE，含有三个独立的外源基因表达盒，分别以VTT及其基因缺失突变株作为原始病毒，以增强型绿色荧光蛋白(EGFP)、红色荧光蛋白（RFP）和蓝色荧光蛋白（BFP）作为报告基因，分别对三个表达盒的功能进行验证。通过噬斑筛选，获得两株重组痘苗病毒(rVTT-EGFP、rdVTT-EGFP)，利用PCR、实时荧光定量PCR、Western blot方法对重组病毒以及对外源基因的表达量和遗传稳定性进行分析。结果表明，成功构建了三基因表达盒痘苗病毒穿梭载体，成功筛选出两株重组痘苗病毒，EGFP基因在痘苗病毒内得到高水平表达，三个表达盒之间没有相互影响，而且重组病毒具有良好的遗传稳定性。 将重组核酸疫苗pVL-CCMp24上的目的免疫原基因CCMp24连接到痘苗病毒穿梭载体pSTKE的第一个表达盒内即MCS1，由特异性引物扩增获得的含Loxp基因序列的EGFP基因（EGFP基因两端的Loxp基因方向相同）克隆到pSTKE的第三个表达盒MCS3中，重组质粒pCCMp24-LEL鉴定正确后，转染dVTT感染的BHK-21细胞，通过同源重组、病毒蚀斑筛选获得重组CCMp24和EGFP的E3L及TK基因缺失的痘苗病毒rddVTT-CCMp24G，然后利用Cre/Loxp重组系统将EGFP基因敲除，获得仅表达目的免疫原基因的双基因缺失重组痘苗病毒rddVTT-CCMp24。RT-PCR、间接免疫荧光、Western Blot检测结果表明，CCMp24融合基因在痘苗病毒感染的BHK-21细胞中得到高效转录和表达。 痘苗病毒在长期的实践应用中具有毒副作用，存在安全隐患。本研究在缺失病毒毒力相关基因的痘苗病毒的基础上，通过痘苗病毒穿梭载体pSTKE携带的目的免疫原基因将TK基因缺失，筛选的基因缺失型重组痘苗病毒通过鼻腔感染和颅内注射感染途径感染BALB/c小鼠，通过监测感染小鼠的体征变化、体重变化、病毒感染后的死亡率等方面评价E3L基因和TK基因缺失的重组痘苗病毒在小鼠体内的毒性。监测结果表明，重组病毒rddVTT-CCMp24的毒力较野生型VTT有很大程度的减弱，提高了进一步研究或应用的安全性。说明缺失E3L基因和TK基因可以使痘苗病毒致弱，且外源基因的引入对病毒的毒力没有显著影响。 在了解其毒力的基础上，以BALB/c小鼠为模型开展了重组病毒rddVTT-CCMp24的免疫原性分析。按照既定的免疫程序免疫后，通过对抗体、细胞因子及T细胞亚型等各项指标的检测，结果显示，rddVTT-CCMp24可诱导机体产生分泌IFN-γ的T细胞数量显著增加，CD4+和CD8+T细胞数增加，脾细胞体外接受HIV特异性抗原刺激下增殖能力显著提高。HIV特异性抗体检测结果显示，可诱导较高的HIV特异性抗体水平，，且诱导IL-2和IL-4细胞因子的分泌。综合结果可知，重组病毒可激发机体细胞和体液免疫应答。 同时，采用DNA/rddVTT-CCMp24prime-boost免疫策略在小鼠模型上检测免疫效果，结果相对于DNA疫苗或rddVTT-CCMp24疫苗分别单独免疫，可更好的刺激分泌IFN-γ产生T细胞的分化和增殖，IFN-γ ELISPOT检测结果显示，HIV特异性的分泌IFN-γ的记忆性T细胞数量显著高于单独免疫组。表明，prime-boost免疫策略诱导较强的免疫应答和免疫记忆形成，为下一步的实验研究提供了数据支持。
[Abstract]:In view of the current epidemic situation of acquired immunodeficiency syndrome (AIDS) and the current status of prevention and treatment, it is urgent to develop a safe and effective AIDS prevention / treatment vaccine. In this study, DNA plasmid and vaccinia virus Tiantan strain were selected as the immunogen delivery system to study the immunogenicity of AIDS vaccine.
In this study, a recombinant nucleic acid vaccine pVL-CCMp24 was constructed by introducing CpG motif and CTB gene as adjuvant on the basis of the HIV multiple epitope gene (MEGNp24) screened by our laboratory for many years. The recombinant DNA plasmid was transfected into 293T cells. The results of RT-PCR and indirect immunofluorescence showed that the recombinant plasmid encoded the target gene. The gene CCMp24 was correctly transcribed and expressed in mammalian cells. BALB/c mice were immunized by intramuscular injection, and the immunogenicity of the recombinant DNA vaccine was analyzed. The results of immune indices such as specific antibodies to HIV, Th1 and Th2 cytokines, T cell subtypes, lymphocyte proliferation and so on showed that the recombinant DNA vaccine pVL-CCMp24 was feasible. To induce a certain degree of cellular and humoral immune response.
Based on the design concept of multivalent vaccine, a vaccinia virus shuttle vector pSTKE containing three expression cassettes was constructed, which contained three independent exogenous gene expression cassettes. VTT and its deleted mutants were used as the original viruses, and enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP) and blue fluorescent protein (B) were used as the primary viruses. Two recombinant vaccinia virus strains (rVTT-EGFP, rdVTT-EGFP) were obtained by plaque screening. The recombinant virus was successfully constructed by PCR, real-time fluorescence quantitative PCR and Western blot. Two recombinant vaccinia viruses were successfully screened out by shuttle vector of three gene expression cassettes. EGFP gene was highly expressed in vaccinia viruses. There was no interaction between the three expression cassettes, and the recombinant virus had good genetic stability.
The target immunogen gene CCMp24 on the recombinant nucleic acid vaccine pVL-CCMp24 was linked to the first expression cassette of vaccinia virus shuttle vector pSTKE, namely, MCS1. The EGFP gene containing Loxp gene sequence (the direction of the Loxp gene at both ends of the EGFP gene) was cloned into the third expression cassette MCS3 of pSTKE by specific primer amplification. CMp24-LEL was identified correctly and transfected into BHK-21 cells infected with dVTT. Recombinant CCMp24, E3L of EGFP and rddVTT-CCMp24G of vaccinia virus with TK gene deletion were obtained by homologous recombination and virus plaque screening. Then, the EGFP gene was knocked out by Cre/Loxp recombinant system, and the recombinant vaccinia virus RDD with double gene deletion was obtained. The results of VTT-CCMp24.RT-PCR, indirect immunofluorescence and Western Blot showed that CCMp24 fusion gene was highly transcribed and expressed in vaccinia virus-infected BHK-21 cells.
Vaccinia virus has toxic side effects and potential safety hazards in its long-term practical application. Based on vaccinia virus with virulence-related genes deleted, TK gene was deleted by the target immunogen gene carried by vaccinia virus shuttle vector pSTKE, and the recombinant vaccinia virus with deleted genes was screened for nose infection and intracranial infection. The toxicity of recombinant vaccinia virus with deletion of E3L gene and TK gene was evaluated in BALB/c mice infected by injection. The results showed that the virulence of recombinant virus rddVTT-CCMp24 was much weaker than that of wild type VTT. The results showed that deletion of E3L gene and TK gene could weaken vaccinia virus, and the introduction of exogenous gene had no significant effect on virulence of the virus.
The immunogenicity of recombinant virus rddVTT-CCMp24 was analyzed in BALB/c mice on the basis of its virulence. The results showed that rddVTT-CCMp24 could induce the production of IFN-gamma-secreting T cells in vivo after immunization according to the established immune program, and the detection of antibodies, cytokines and T cell subtypes. The results showed that the recombinant virus could induce high levels of HIV-specific antibodies and secretion of IL-2 and IL-4 cytokines. The results showed that the recombinant virus could stimulate cellular and humoral immunity. Answer.
At the same time, DNA/rddVTT-CCMp24 prime-boost immunization strategy was used to detect the immune effect in mice model. Results Compared with DNA vaccine or rddVTT-CCMp24 vaccine alone, it could stimulate the differentiation and proliferation of T cells secreting IFN-gamma better. The results of IFN-gamma ELISPOT showed that the memory T cells secreting IFN-gamma were specific to HIV. The results showed that prime-boost strategy could induce stronger immune response and immune memory formation, which provided data support for further experimental study.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2236511
[Abstract]:In view of the current epidemic situation of acquired immunodeficiency syndrome (AIDS) and the current status of prevention and treatment, it is urgent to develop a safe and effective AIDS prevention / treatment vaccine. In this study, DNA plasmid and vaccinia virus Tiantan strain were selected as the immunogen delivery system to study the immunogenicity of AIDS vaccine.
In this study, a recombinant nucleic acid vaccine pVL-CCMp24 was constructed by introducing CpG motif and CTB gene as adjuvant on the basis of the HIV multiple epitope gene (MEGNp24) screened by our laboratory for many years. The recombinant DNA plasmid was transfected into 293T cells. The results of RT-PCR and indirect immunofluorescence showed that the recombinant plasmid encoded the target gene. The gene CCMp24 was correctly transcribed and expressed in mammalian cells. BALB/c mice were immunized by intramuscular injection, and the immunogenicity of the recombinant DNA vaccine was analyzed. The results of immune indices such as specific antibodies to HIV, Th1 and Th2 cytokines, T cell subtypes, lymphocyte proliferation and so on showed that the recombinant DNA vaccine pVL-CCMp24 was feasible. To induce a certain degree of cellular and humoral immune response.
Based on the design concept of multivalent vaccine, a vaccinia virus shuttle vector pSTKE containing three expression cassettes was constructed, which contained three independent exogenous gene expression cassettes. VTT and its deleted mutants were used as the original viruses, and enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP) and blue fluorescent protein (B) were used as the primary viruses. Two recombinant vaccinia virus strains (rVTT-EGFP, rdVTT-EGFP) were obtained by plaque screening. The recombinant virus was successfully constructed by PCR, real-time fluorescence quantitative PCR and Western blot. Two recombinant vaccinia viruses were successfully screened out by shuttle vector of three gene expression cassettes. EGFP gene was highly expressed in vaccinia viruses. There was no interaction between the three expression cassettes, and the recombinant virus had good genetic stability.
The target immunogen gene CCMp24 on the recombinant nucleic acid vaccine pVL-CCMp24 was linked to the first expression cassette of vaccinia virus shuttle vector pSTKE, namely, MCS1. The EGFP gene containing Loxp gene sequence (the direction of the Loxp gene at both ends of the EGFP gene) was cloned into the third expression cassette MCS3 of pSTKE by specific primer amplification. CMp24-LEL was identified correctly and transfected into BHK-21 cells infected with dVTT. Recombinant CCMp24, E3L of EGFP and rddVTT-CCMp24G of vaccinia virus with TK gene deletion were obtained by homologous recombination and virus plaque screening. Then, the EGFP gene was knocked out by Cre/Loxp recombinant system, and the recombinant vaccinia virus RDD with double gene deletion was obtained. The results of VTT-CCMp24.RT-PCR, indirect immunofluorescence and Western Blot showed that CCMp24 fusion gene was highly transcribed and expressed in vaccinia virus-infected BHK-21 cells.
Vaccinia virus has toxic side effects and potential safety hazards in its long-term practical application. Based on vaccinia virus with virulence-related genes deleted, TK gene was deleted by the target immunogen gene carried by vaccinia virus shuttle vector pSTKE, and the recombinant vaccinia virus with deleted genes was screened for nose infection and intracranial infection. The toxicity of recombinant vaccinia virus with deletion of E3L gene and TK gene was evaluated in BALB/c mice infected by injection. The results showed that the virulence of recombinant virus rddVTT-CCMp24 was much weaker than that of wild type VTT. The results showed that deletion of E3L gene and TK gene could weaken vaccinia virus, and the introduction of exogenous gene had no significant effect on virulence of the virus.
The immunogenicity of recombinant virus rddVTT-CCMp24 was analyzed in BALB/c mice on the basis of its virulence. The results showed that rddVTT-CCMp24 could induce the production of IFN-gamma-secreting T cells in vivo after immunization according to the established immune program, and the detection of antibodies, cytokines and T cell subtypes. The results showed that the recombinant virus could induce high levels of HIV-specific antibodies and secretion of IL-2 and IL-4 cytokines. The results showed that the recombinant virus could stimulate cellular and humoral immunity. Answer.
At the same time, DNA/rddVTT-CCMp24 prime-boost immunization strategy was used to detect the immune effect in mice model. Results Compared with DNA vaccine or rddVTT-CCMp24 vaccine alone, it could stimulate the differentiation and proliferation of T cells secreting IFN-gamma better. The results of IFN-gamma ELISPOT showed that the memory T cells secreting IFN-gamma were specific to HIV. The results showed that prime-boost strategy could induce stronger immune response and immune memory formation, which provided data support for further experimental study.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【引证文献】
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1 刘存霞;杜寿文;李昌;王宇航;王茂鹏;李沂;尹荣兰;李霄;任大勇;秦艳青;任静强;金宁一;;HIV复合表位DNA重组体和痘苗病毒疫苗联合免疫实验研究[J];中国科学:生命科学;2013年05期
本文编号：2236511
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