佐劑對(duì)HIV-1 gp120 DNA疫苗免疫效果的影響
發(fā)布時(shí)間：2018-10-08 21:19
【摘要】：艾滋�。ˋcquired Immune Deficiency Syndrome，AIDS）又稱獲得性免疫缺陷綜合征，是由人類免疫缺陷病毒（Human Immunodeficiency Virus，HIV）引起的一種傳染性及病死率非常高的疾病，世界上每年大約有200多萬人死于艾滋病，感染人群在全世界分布極為廣泛。在研制的各種HIV疫苗中，普遍認(rèn)為DNA疫苗是一種具有發(fā)展?jié)摿Φ男滦鸵呙�，因�(yàn)樗杀恢苯愚D(zhuǎn)染進(jìn)機(jī)體細(xì)胞內(nèi)并且表達(dá)抗原蛋白，這樣減少了體外進(jìn)行蛋白表達(dá)和純化的過程；質(zhì)粒DNA結(jié)構(gòu)簡單，提純質(zhì)粒工藝簡便，適合大規(guī)模生產(chǎn)。雖然DNA疫苗存在很多優(yōu)勢，但是HIV DNA疫苗的臨床試驗(yàn)還沒有成功的例子，主要原因即為HIV DNA疫苗的免疫原性較差，如何提高DNA疫苗的免疫效果仍是亟待解決的問題。近年來，對(duì)DNA疫苗的研究實(shí)驗(yàn)結(jié)果表明，佐劑可以顯著提高DNA疫苗在機(jī)體內(nèi)的免疫原性，增強(qiáng)疫苗的免疫效果。在本課題中，我們用HIV-1gp120DNA疫苗與實(shí)驗(yàn)室現(xiàn)有的一些佐劑聯(lián)合免疫BALB/c小鼠，用間接ELISA法檢測小鼠血清中抗gp120特異性IgG抗體水平，評(píng)價(jià)不同佐劑對(duì)HIV-1gp120DNA疫苗免疫效果的影響。 在本實(shí)驗(yàn)中所用的HIV-1gp120DNA疫苗為中國疾病控制中心（CDC）構(gòu)建的兩種質(zhì)粒pENVPOL (簡稱EP)和pGAGTNR(簡稱GT)，為高純度質(zhì)粒DNA，不能含有宿主DNA，RNA以及蛋白質(zhì)等雜質(zhì)。而在實(shí)驗(yàn)室廣泛應(yīng)用的提取質(zhì)粒DNA的方法為傳統(tǒng)堿裂解法，這種方法主要采用酚氯仿等有機(jī)試劑抽提蛋白質(zhì)以及RNA酶消化RNA，最終所提取的質(zhì)粒DNA無法滿足動(dòng)物注射級(jí)應(yīng)用的DNA疫苗的標(biāo)準(zhǔn)。在實(shí)驗(yàn)室現(xiàn)有的條件下，我們?nèi)绾潍@取制備DNA疫苗的工藝方法是迫切解決的問題。在本課題研究的第一部分即為摸索一種經(jīng)濟(jì)、簡便的方法從而大量制備符合動(dòng)物實(shí)驗(yàn)應(yīng)用的DNA疫苗。首先嘗試了一些簡便的提取質(zhì)粒的實(shí)驗(yàn)操作，這些操作都是在堿裂解法的基礎(chǔ)上進(jìn)行改進(jìn)的，，通過各種實(shí)驗(yàn)方法的比較最終獲得了一種最為有效的提取質(zhì)粒DNA的方法。該方法主要為傳統(tǒng)堿裂解法與氯化鈣、PEG8000試劑相結(jié)合提取質(zhì)粒DNA，氯化鈣可選擇性沉淀質(zhì)粒中的大量RNA，PEG8000可選擇性沉淀超螺旋質(zhì)粒DNA。此方法大大降低了質(zhì)粒中蛋白質(zhì)、宿主RNA、基因組DNA、有機(jī)物質(zhì)等雜質(zhì)的殘留。將所提取的質(zhì)粒粗提液通過Q Sepharose F.F.強(qiáng)陰離子交換柱去除內(nèi)毒素，最終制備了約7mg的HIV-1gp120雙質(zhì)粒DNA(內(nèi)毒素0.05EU/μg，濃度為2μg/μl)。經(jīng)各項(xiàng)檢測指標(biāo)判斷，質(zhì)粒DNA的純度以及含量均已滿足后續(xù)動(dòng)物實(shí)驗(yàn)應(yīng)用的要求，并且制備DNA疫苗的成本低廉，操作簡便，適合實(shí)驗(yàn)室應(yīng)用。 大量實(shí)驗(yàn)證明佐劑能夠增強(qiáng)DNA疫苗的免疫原性，我們選用佐劑MF59、CpGODN、WFR聯(lián)合EP+GT（質(zhì)粒DNA）免疫BALB/c小鼠，隨機(jī)分為4組：EP+GT；MF59+EP+GT；CpG ODN+MF59+EP+GT；WFR+EP+GT。每組6只小鼠，采用脛前肌肉注射的方式免疫，一共免疫三次，每次間隔2周。分別在不同時(shí)間點(diǎn)取小鼠血清，用間接ELISA方法檢測小鼠血清中抗gp120特異性IgG體液免疫應(yīng)答，結(jié)果表明應(yīng)用WFR佐劑組的小鼠體液免疫應(yīng)答水平與其他各組相比效果較好。在初次免疫后的56天用MTT法檢測WFR+EP+GT組、EP+GT組及空白對(duì)照組小鼠脾細(xì)胞增殖能力，結(jié)果表明，gp120多肽對(duì)各組小鼠脾細(xì)胞均有刺激作用，用終濃度為1μg/ml gp120多肽比用10μg/ml gp120多肽更易刺激體外脾細(xì)胞的增殖情況，但是并無統(tǒng)計(jì)學(xué)差異。在有或無gp120多肽刺激的條件下，EP+GT+WFR組小鼠脾細(xì)胞的增殖程度均大于EP+GT組小鼠及健康空白小鼠的脾細(xì)胞增殖程度，具有統(tǒng)計(jì)學(xué)意義。用PFR佐劑免疫小鼠，隨機(jī)分為2組：EP+GT，PFR+EP+GT，每組7只，分三次脛前肌肉注射免疫，間隔2周，分別在不同時(shí)間點(diǎn)檢測小鼠體內(nèi)針對(duì)gp120多肽的特異IgG體液免疫應(yīng)答。結(jié)果發(fā)現(xiàn)PFR+EP+GT組的小鼠與單獨(dú)應(yīng)用質(zhì)粒組的小鼠相比對(duì)抗原刺激產(chǎn)生更顯著的體液免疫應(yīng)答，并且在免疫后87天兩組小鼠血清中抗gp120抗體仍然維持較高水平。 本研究主要通過對(duì)提取質(zhì)粒方法學(xué)的摸索，獲得了一種制備動(dòng)物實(shí)驗(yàn)應(yīng)用的DNA疫苗的簡便方法。將不同佐劑與HIV-1gp120DNA疫苗聯(lián)合應(yīng)用，結(jié)果表明WFR、PFR佐劑具有增強(qiáng)DNA疫苗免疫原性的作用，為進(jìn)一步對(duì)該佐劑的深入研究提供了實(shí)驗(yàn)基礎(chǔ)。
[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,
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
本文編號(hào)：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,
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 戴波濤;趙剛;;獸用疫苗佐劑研究現(xiàn)狀[J];現(xiàn)代畜牧獸醫(yī);2013年02期
本文編號(hào)：2258300
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