佐剂对HIV-1 gp120 DNA疫苗免疫效果的影响
发布时间：2018-10-08 21:19
【摘要】：艾滋病（Acquired Immune Deficiency Syndrome，AIDS）又称获得性免疫缺陷综合征，是由人类免疫缺陷病毒（Human Immunodeficiency Virus，HIV）引起的一种传染性及病死率非常高的疾病，世界上每年大约有200多万人死于艾滋病，感染人群在全世界分布极为广泛。在研制的各种HIV疫苗中，普遍认为DNA疫苗是一种具有发展潜力的新型疫苗，因为它可被直接转染进机体细胞内并且表达抗原蛋白，这样减少了体外进行蛋白表达和纯化的过程；质粒DNA结构简单，提纯质粒工艺简便，适合大规模生产。虽然DNA疫苗存在很多优势，但是HIV DNA疫苗的临床试验还没有成功的例子，主要原因即为HIV DNA疫苗的免疫原性较差，如何提高DNA疫苗的免疫效果仍是亟待解决的问题。近年来，对DNA疫苗的研究实验结果表明，佐剂可以显著提高DNA疫苗在机体内的免疫原性，增强疫苗的免疫效果。在本课题中，我们用HIV-1gp120DNA疫苗与实验室现有的一些佐剂联合免疫BALB/c小鼠，用间接ELISA法检测小鼠血清中抗gp120特异性IgG抗体水平，评价不同佐剂对HIV-1gp120DNA疫苗免疫效果的影响。 在本实验中所用的HIV-1gp120DNA疫苗为中国疾病控制中心（CDC）构建的两种质粒pENVPOL (简称EP)和pGAGTNR(简称GT)，为高纯度质粒DNA，不能含有宿主DNA，RNA以及蛋白质等杂质。而在实验室广泛应用的提取质粒DNA的方法为传统碱裂解法，这种方法主要采用酚氯仿等有机试剂抽提蛋白质以及RNA酶消化RNA，最终所提取的质粒DNA无法满足动物注射级应用的DNA疫苗的标准。在实验室现有的条件下，我们如何获取制备DNA疫苗的工艺方法是迫切解决的问题。在本课题研究的第一部分即为摸索一种经济、简便的方法从而大量制备符合动物实验应用的DNA疫苗。首先尝试了一些简便的提取质粒的实验操作，这些操作都是在碱裂解法的基础上进行改进的，，通过各种实验方法的比较最终获得了一种最为有效的提取质粒DNA的方法。该方法主要为传统碱裂解法与氯化钙、PEG8000试剂相结合提取质粒DNA，氯化钙可选择性沉淀质粒中的大量RNA，PEG8000可选择性沉淀超螺旋质粒DNA。此方法大大降低了质粒中蛋白质、宿主RNA、基因组DNA、有机物质等杂质的残留。将所提取的质粒粗提液通过Q Sepharose F.F.强阴离子交换柱去除内毒素，最终制备了约7mg的HIV-1gp120双质粒DNA(内毒素0.05EU/μg，浓度为2μg/μl)。经各项检测指标判断，质粒DNA的纯度以及含量均已满足后续动物实验应用的要求，并且制备DNA疫苗的成本低廉，操作简便，适合实验室应用。 大量实验证明佐剂能够增强DNA疫苗的免疫原性，我们选用佐剂MF59、CpGODN、WFR联合EP+GT（质粒DNA）免疫BALB/c小鼠，随机分为4组：EP+GT；MF59+EP+GT；CpG ODN+MF59+EP+GT；WFR+EP+GT。每组6只小鼠，采用胫前肌肉注射的方式免疫，一共免疫三次，每次间隔2周。分别在不同时间点取小鼠血清，用间接ELISA方法检测小鼠血清中抗gp120特异性IgG体液免疫应答，结果表明应用WFR佐剂组的小鼠体液免疫应答水平与其他各组相比效果较好。在初次免疫后的56天用MTT法检测WFR+EP+GT组、EP+GT组及空白对照组小鼠脾细胞增殖能力，结果表明，gp120多肽对各组小鼠脾细胞均有刺激作用，用终浓度为1μg/ml gp120多肽比用10μg/ml gp120多肽更易刺激体外脾细胞的增殖情况，但是并无统计学差异。在有或无gp120多肽刺激的条件下，EP+GT+WFR组小鼠脾细胞的增殖程度均大于EP+GT组小鼠及健康空白小鼠的脾细胞增殖程度，具有统计学意义。用PFR佐剂免疫小鼠，随机分为2组：EP+GT，PFR+EP+GT，每组7只，分三次胫前肌肉注射免疫，间隔2周，分别在不同时间点检测小鼠体内针对gp120多肽的特异IgG体液免疫应答。结果发现PFR+EP+GT组的小鼠与单独应用质粒组的小鼠相比对抗原刺激产生更显著的体液免疫应答，并且在免疫后87天两组小鼠血清中抗gp120抗体仍然维持较高水平。 本研究主要通过对提取质粒方法学的摸索，获得了一种制备动物实验应用的DNA疫苗的简便方法。将不同佐剂与HIV-1gp120DNA疫苗联合应用，结果表明WFR、PFR佐剂具有增强DNA疫苗免疫原性的作用，为进一步对该佐剂的深入研究提供了实验基础。
[Abstract]:Acquired Immune Deficiency Syndrome (AIDS), also known as Acquired Immune Deficiency Syndrome (AIDS), is an infectious and high-mortality disease caused by human immunodeficiency virus (HIV). More than 2 million people die in AIDS every year in the world. The infected population is widely distributed throughout the world. In the development of various HIV vaccines, it is generally believed that DNA vaccine is a new type of vaccine with development potential, because it can be directly transfected into the body cell and expresses the antigen protein, thus reducing the process of protein expression and purification in vitro, and the plasmid DNA structure is simple, The purification plasmid process is simple and convenient, and is suitable for large-scale production. Although DNA vaccine has many advantages, the clinical trial of HIV DNA vaccine has not been successful. The main reason is that the immunogenicity of HIV DNA vaccine is poor, and how to improve the immune effect of DNA vaccine is still a problem to be solved. In recent years, the experimental results of DNA vaccine show that adjuvant can significantly improve the immunogenicity of DNA vaccine in organism and enhance the immune effect of vaccine. In this study, we immunized BALB/ c mice with HIV-1gp120DNA vaccine and some existing adjuvants in our laboratory, and tested the anti-gp120 specific IgG antibody level in serum of mice by indirect ELISA, and evaluated the effect of different adjuvant on the immune effect of HIV-1gp120DNA vaccine. The HIV-1gp120DNA vaccine used in the experiment is the two plasmids pENVPOL (hereinafter referred to as EP) and pGAGTNR (hereinafter referred to as GT) constructed by China Disease Control Center (CDC), which are high-purity plasmid DNA and can not contain host DNA, RNA and protein, etc. The method for extracting plasmid DNA widely used in the laboratory is a traditional alkaline lysis method, which mainly adopts organic reagent extraction protein such as phenol chloroform and RNA enzyme digestion RNA, and finally the extracted plasmid DNA can not meet the DNA vaccine of the animal injection grade application. Standard. How to get a DNA vaccine is urgently addressed under existing conditions in the lab The first part of this study is to find a kind of economical and simple method, so as to produce a lot of DNA which accords with the animal experiment application. First of all, we tried some simple experiments to extract the plasmid, which were improved on the basis of the alkaline lysis method, and the most effective extraction plasmid DNA was obtained through the comparison of various experimental methods. The method is mainly used for extracting plasmid DNA with the combination of the traditional alkali cracking method and the calcium chloride and the PEG8000 reagent, the calcium chloride can selectively precipitate a large amount of RNA in the plasmid, the PEG8000 can selectively precipitate the supercoiled plasmid D, NA. The method greatly reduces the impurities such as protein, host RNA, genomic DNA, organic matter and the like in the plasmid Residual. The extracted plasmid crude extract was removed by Q Sepharose F. F. Strong anion exchange column to finally prepare about 7mg of HIV-1gp120 double plasmid DNA (endotoxin 0. 05EU/. mu.g, with a concentration of 2. mu.g/. and 1) judging whether the purity and the content of the plasmid DNA meet the requirements of the follow-up animal experimental application after the detection indexes are judged, A lot of experiments prove that adjuvant can enhance the immunogenicity of DNA vaccine. We use adjuvant MF59, CpGODN, WFR and EP + GT (plasmid DNA) to immunize BALB/ c mice, randomly divided into 4 groups: EP + GT; MF59 + EP + GT; CpG island + MF59 + EP + GT; WFR + EP + GT. Six mice in each group were immunized with intramuscular injection of the tibia for three times each. Mice serum were taken at different time points, and the immune response of anti-gp120 specific IgG was detected by indirect ELISA. The results showed that the level of humoral immune response in mice treated with WFR adjuvant group was compared with other groups. The proliferation ability of spleen cells in WFR + EP + GT group, EP + GT group and blank control group was detected by MTT assay for 56 days after initial immunization. The results showed that the gp120 polypeptide had no effect on spleen cells in each group. To stimulate the proliferation of in vitro spleen cells with a final concentration of 1. m u.g/ ml gp120 polypeptide than 10. m u.g/ ml of gp120 polypeptides, but not The proliferative degree of splenocytes in EP + GT + WFR group was higher than that of EP + GT group and healthy blank mice under the condition of stimulation with or without gp120 polypeptide. The mice were immunized with PFR adjuvant and randomly divided into 2 groups: EP + GT, PFR + EP + GT, 7 rabbits in each group, three times of pre-tibial muscle injection immunization, 2 weeks interval, and the specific IgG antibody against gp120 polypeptide was detected in mice at different time points. The results showed that mice in the PFR + EP + GT group had a more significant humoral immune response compared to mice treated with the plasmid group alone, and the anti-gp120 antibody in the sera of the two groups after immunization was still dimension In this study, a new method for the preparation of animal experiment was obtained by exploring the method of extracting plasmid. A simple and convenient method for vaccine preparation is characterized in that different adjuvants are combined with HIV-1gp120DNA vaccine, and the results show that WFR and PFR adjuvant have the effect of enhancing the immunogenicity of DNA vaccine,
【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：2258300
[Abstract]:Acquired Immune Deficiency Syndrome (AIDS), also known as Acquired Immune Deficiency Syndrome (AIDS), is an infectious and high-mortality disease caused by human immunodeficiency virus (HIV). More than 2 million people die in AIDS every year in the world. The infected population is widely distributed throughout the world. In the development of various HIV vaccines, it is generally believed that DNA vaccine is a new type of vaccine with development potential, because it can be directly transfected into the body cell and expresses the antigen protein, thus reducing the process of protein expression and purification in vitro, and the plasmid DNA structure is simple, The purification plasmid process is simple and convenient, and is suitable for large-scale production. Although DNA vaccine has many advantages, the clinical trial of HIV DNA vaccine has not been successful. The main reason is that the immunogenicity of HIV DNA vaccine is poor, and how to improve the immune effect of DNA vaccine is still a problem to be solved. In recent years, the experimental results of DNA vaccine show that adjuvant can significantly improve the immunogenicity of DNA vaccine in organism and enhance the immune effect of vaccine. In this study, we immunized BALB/ c mice with HIV-1gp120DNA vaccine and some existing adjuvants in our laboratory, and tested the anti-gp120 specific IgG antibody level in serum of mice by indirect ELISA, and evaluated the effect of different adjuvant on the immune effect of HIV-1gp120DNA vaccine. The HIV-1gp120DNA vaccine used in the experiment is the two plasmids pENVPOL (hereinafter referred to as EP) and pGAGTNR (hereinafter referred to as GT) constructed by China Disease Control Center (CDC), which are high-purity plasmid DNA and can not contain host DNA, RNA and protein, etc. The method for extracting plasmid DNA widely used in the laboratory is a traditional alkaline lysis method, which mainly adopts organic reagent extraction protein such as phenol chloroform and RNA enzyme digestion RNA, and finally the extracted plasmid DNA can not meet the DNA vaccine of the animal injection grade application. Standard. How to get a DNA vaccine is urgently addressed under existing conditions in the lab The first part of this study is to find a kind of economical and simple method, so as to produce a lot of DNA which accords with the animal experiment application. First of all, we tried some simple experiments to extract the plasmid, which were improved on the basis of the alkaline lysis method, and the most effective extraction plasmid DNA was obtained through the comparison of various experimental methods. The method is mainly used for extracting plasmid DNA with the combination of the traditional alkali cracking method and the calcium chloride and the PEG8000 reagent, the calcium chloride can selectively precipitate a large amount of RNA in the plasmid, the PEG8000 can selectively precipitate the supercoiled plasmid D, NA. The method greatly reduces the impurities such as protein, host RNA, genomic DNA, organic matter and the like in the plasmid Residual. The extracted plasmid crude extract was removed by Q Sepharose F. F. Strong anion exchange column to finally prepare about 7mg of HIV-1gp120 double plasmid DNA (endotoxin 0. 05EU/. mu.g, with a concentration of 2. mu.g/. and 1) judging whether the purity and the content of the plasmid DNA meet the requirements of the follow-up animal experimental application after the detection indexes are judged, A lot of experiments prove that adjuvant can enhance the immunogenicity of DNA vaccine. We use adjuvant MF59, CpGODN, WFR and EP + GT (plasmid DNA) to immunize BALB/ c mice, randomly divided into 4 groups: EP + GT; MF59 + EP + GT; CpG island + MF59 + EP + GT; WFR + EP + GT. Six mice in each group were immunized with intramuscular injection of the tibia for three times each. Mice serum were taken at different time points, and the immune response of anti-gp120 specific IgG was detected by indirect ELISA. The results showed that the level of humoral immune response in mice treated with WFR adjuvant group was compared with other groups. The proliferation ability of spleen cells in WFR + EP + GT group, EP + GT group and blank control group was detected by MTT assay for 56 days after initial immunization. The results showed that the gp120 polypeptide had no effect on spleen cells in each group. To stimulate the proliferation of in vitro spleen cells with a final concentration of 1. m u.g/ ml gp120 polypeptide than 10. m u.g/ ml of gp120 polypeptides, but not The proliferative degree of splenocytes in EP + GT + WFR group was higher than that of EP + GT group and healthy blank mice under the condition of stimulation with or without gp120 polypeptide. The mice were immunized with PFR adjuvant and randomly divided into 2 groups: EP + GT, PFR + EP + GT, 7 rabbits in each group, three times of pre-tibial muscle injection immunization, 2 weeks interval, and the specific IgG antibody against gp120 polypeptide was detected in mice at different time points. The results showed that mice in the PFR + EP + GT group had a more significant humoral immune response compared to mice treated with the plasmid group alone, and the anti-gp120 antibody in the sera of the two groups after immunization was still dimension In this study, a new method for the preparation of animal experiment was obtained by exploring the method of extracting plasmid. A simple and convenient method for vaccine preparation is characterized in that different adjuvants are combined with HIV-1gp120DNA vaccine, and the results show that WFR and PFR adjuvant have the effect of enhancing the immunogenicity of DNA vaccine,
【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】
相关期刊论文 前1条
1 戴波涛;赵刚;;兽用疫苗佐剂研究现状[J];现代畜牧兽医;2013年02期
本文编号：2258300
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