重组疟疾疫苗PfCP-2.9单克隆抗体的特性分析
发布时间：2018-11-20 05:44
【摘要】： 疟疾是世界上分布和流行最广、危害最为严重的寄生虫性传染病之一,世界上有20亿人口生活在疫区。由于疟原虫抗药性以及蚊媒抗杀虫剂抗性的产生和扩散,这给疟疾防治带来了很大的困难。尽管疟疾疫苗研制也面临疟原虫生活史复杂和抗原变异等困难,但研制有效的疟疾疫苗仍是人们期望能用于控制疟疾的潜在新途径。 恶性疟原虫主要裂殖子表面抗原1(MSP1)19kD C-末端区域(MSP1-19)与裂殖子顶端膜抗原1的第三功能域(AMA-1(III))均是裂殖子入侵红细胞时被酶解的片段,在裂殖子入侵过程中发挥重要作用,已成为疟原虫红内期疫苗的两个重要候选抗原。本实验室将MSP1-19和AMA-1(III)两个抗原通过一段链接区序列(由28个氨基酸残基组成的多肽,定名为P28)进行融合,形成融合抗原,即PfCP-2.9融合蛋白。该融合抗原作为疟疾疫苗候选抗原已完成2个临床试验。与国外同类疫苗相比,此种PfCP-2.9重组疟疾疫苗已显示出明显的优点:①高免疫原性和强抑制疟原虫生长的效力。与两个抗原成分相比,抗体滴度分别增加22和47倍;融合抗原免疫原性增强使其免疫血清在15%浓度时能完全抑制疟原虫生长,而单个抗原免疫或将两个抗原混合后免疫,其免疫血清在15%浓度时对疟原虫生长无明显抑制作用;②不同地区疟疾病人血清对PfCP-2.9融合抗原的免疫反应性比单抗原成分明显增强;③在毕氏酵母分泌表达系统中,PfCP-2.9融合抗原表达产量达到2.6克/升,比单抗原在同一系统中表达产量高约20倍,且可溶性和稳定性明显提高。 本实验室在前期研究中采用PfCP-2.9重组蛋白制备了一组单克隆抗体,主要用于PfCP-2.9疫苗免疫保护作用的机制探讨,包括PfCP-2.9融合抗原的表位鉴定,通过与PfCP-2.9疫苗接种者免疫血清的竞争ELISA试验,分析该疫苗的一些重要表位在人体是否得到有效递呈,产生免疫应答等。这些研究预期结果将部分阐明PfCP-2.9融合抗原疫苗的免疫保护作用机制,并为融合抗原疫苗的设计提供依据和信息。 本研究对21株PfCP-2.9融合抗原单克隆抗体的特性进行分析。通过杂交瘤细胞的培养和BALB/c小鼠腹腔注射制备一定量的单克隆抗体,在此基础上开展以下的研究并取得结果如下:(1)单抗与融合蛋白的反应性:用ELISA检测21株单抗,结果为阳性反应的18株,另外3株为阴性;用Western blot方法检测结果:同样为18株阳性;(2)单克隆抗体识别表位构象分析:将PfCP-2.9抗原用尿素进行彻底变性,再用单抗与变性和未变性PfCP-2.9蛋白进行Western blot反应,结果显示其中6株可以识别变性和未变性蛋白,表明这些单抗识别的是线性表位;而剩下的12株单抗不识别变性后的PfCP-2.9融合抗原,表明这些单抗识别的是构象表位;(3)单抗识别区域的分析:以本实验室制备的MSP1-19和美国NIH提供的AMA-1标准品为抗原,用Western blot的方法对18株单抗的特异性识别区域进行检测,结果为识别MSP1-19的单抗共5株,而识别AMA-1的单抗有8株,剩余的5株单抗既不识别MSP1-19,也不识别AMA-1,但识别PfCP-2.9融合蛋白;(4)表位竞争性分析:以PfCP-2.9为抗原,用竞争ELISA方法先后用过量未标记单抗和HRP标记单抗与之反应,结果有4对单抗出现相互竞争,提示这4对单抗可能识别同一表位或者是识别的表位在构象上或者在识别序列上存在相互影响;(5)单克隆抗体与疟原虫天然蛋白反应性:以处于成熟裂殖体期的恶性疟原虫(FCC1/HN株)为抗原,以血涂片的形式用间接免疫荧光方法(IFA)对单克隆抗体的生物学活性进行测定。结果显示:有8株单抗能够不同程度的与疟原虫天然虫体反应。(6)单抗体外抑制疟原虫生长的效力分析:在红细胞压积到2%,起始原虫率为0.5%的体系中,采用LDH方法分别测定各单抗抑制疟原虫生长的效力。结果表明:当单抗终浓度为0.5mg/ml时,有6株单抗不同程度抑制疟原虫生长。 本研究对PfCP-2.9重组蛋白的一组单克隆抗体的特性进行了初步分析。本研究的结果为开展PfCP-2.9疟疾疫苗免疫保护作用机制研究提供重要信息和基础,继而为设计和完善融合抗原疟疾疫苗提供依据。
[Abstract]:Malaria is one of the most widespread and most harmful parasitic infectious diseases in the world, with a population of 2 billion in the world living in the epidemic area. Because of the drug resistance of the plasmodium and the generation and diffusion of the anti-insecticide resistance of the mosquito-borne, this brings great difficulty to the prevention and control of malaria. The development of malaria vaccine is also a potential new way for malaria to be used to control malaria, although the development of malaria vaccine is also faced with the difficulties of complex and antigenic variation of the plasmodium. The third functional domain (AMA-1 (III)) of the main merozoite surface antigen 1 (MSP1) 19kD C-terminal region (MSP1-19) and the merozoite top membrane antigen 1 of the plasmodium falciparum are the fragments of the enzyme solution when the merozoites invade red blood cells and play a role in the process of the invasion of merozoites. It's an important role, and it has become two important parts of the plasmodium's red-phase vaccine. candidate antigens. The laboratory fusion of two antigens of MSP1-19 and AMA-1 (III) through a sequence of linked regions (a polypeptide consisting of 28 amino acid residues, designated P28) to form a fusion antigen, i.e., PfCP-2.9. Fusion protein. The fusion antigen has been completed as a candidate antigen for the malaria vaccine Clinical trials. The PfCP-2. 9 recombinant malaria vaccine has shown significant advantages over foreign similar vaccines: high immunogenicity and strong inhibition of plasmodium Long efficacy. The antibody drop is increased by 22 and 47 fold, respectively, compared to the two antigen components; the fusion antigen immunogenicity enhances the ability of the immune serum to completely inhibit the growth of the parasite at a concentration of 15%, while the individual antigen is immunized or mixed with two antigens The immune response of PfCP-2. 9 fusion antigen was higher than that of single antigen. In the expression system of Pichia pastoris, the expression of PfCP-2.9 fusion antigen was 2. 6 g/ l, which is about 20 times higher than that of a single antigen in the same system, and is soluble and stable In this lab, a group of monoclonal antibodies were prepared by using the PfCP-2.9 recombinant protein in the earlier study, which was mainly used in the mechanism of the immune protection of the PfCP-2.9 vaccine, including the surface identification of the PfCP-2.9 fusion antigen, and the immune serum with the PfCP-2.9 vaccine. The competitive ELISA test is used to analyze whether some important table bits of the vaccine are effectively delivered to the human body The results of these studies will partially illustrate the mechanism of the immune protection of the PfCP-2. 9 fusion antigen vaccine and provide a vaccine for the fusion of the antigen. The basis and information of the design were provided. The 21 strains of PfCP-2.9 were fused in this study. The characteristics of the monoclonal antibody of the antigen were analyzed. A certain amount of the monoclonal antibody was prepared by the cultivation of the hybridoma cells and the intraperitoneal injection of BALB/ c mice. The following studies were carried out and the results are as follows: (1) the reactivity of the monoclonal antibody and the fusion protein: 21 strains were detected by ELISA The results were positive, and the other 3 strains were negative. The results were detected by Western blot. The results were as follows: 18 strains were positive; (2) the identification of the monoclonal antibody was carried out: the PfCP-2.9 antigen was completely denatured with urea, and then the protein was added with the modified and undenatured PfCP-2.9protein. Western blot showed that 6 of them can recognize the denatured and undenatured protein, indicating that these monoclonal antibodies recognize the linear table position, while the remaining 12 monoclonal antibodies do not recognize the modified PfCP-2.9 fusion antigen, indicating that these monoclonal antibodies recognize the conformation table. Location: (3) Analysis of the identification area of the monoclonal antibody: The MSP1-19 prepared in the laboratory and the AMA-1 standard provided by the NIH are the antigen, and the specific identification area of the 18 monoclonal antibodies is detected by Western blot. The result is that 5 strains of the monoclonal antibody of MSP1-19 are identified, 8 of the monoclonal antibodies to the AMA-1 are identified, and the remaining 5 Both MSP1-19 and AMA-1 were not identified, but the PfCP-2. 9 fusion protein was identified. (4) Table-position competitive assay: PfCP-2.9 was used as the antigen, and the antibody and HRP-labeled monoclonal antibody were used in the competitive ELISA. The results show that 4 pairs of monoclonal antibodies are competing with each other, suggesting that the 4 pairs of monoclonal antibodies may identify the same table or that the identified table bits have an interaction with the conformation or in the identification sequence; and (5) the reactivity of the monoclonal antibody against the natural protein of the plasmodium: The worm (FCC1/ HN strain) is an antigen and is used in the form of a blood smear by an indirect immunofluorescence method (IFA) The biological activity of the cloned antibody was determined. The results showed that 8 monoclonal antibodies could not The effect of (6) single-antibody on the growth of plasmodium falciparum was studied. The results showed that in the system with 2% red blood cells and 0.5% of the initial protozoa, the LDH method was used to measure the growth of plasmodium. The effect of monoclonal antibody on the growth of plasmodium was determined. The results showed that when the final concentration of the monoclonal antibody was 0.5mg/ ml, there were 6. The growth of plasmodium was inhibited by the different degree of monoclonal antibody. The recombinant eggs of PfCP-2.9were studied in this study. The results of this study provide important information and foundation for the study on the mechanism of the immune protection of the PfCP-2.9malaria vaccine
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
[Abstract]:Malaria is one of the most widespread and most harmful parasitic infectious diseases in the world, with a population of 2 billion in the world living in the epidemic area. Because of the drug resistance of the plasmodium and the generation and diffusion of the anti-insecticide resistance of the mosquito-borne, this brings great difficulty to the prevention and control of malaria. The development of malaria vaccine is also a potential new way for malaria to be used to control malaria, although the development of malaria vaccine is also faced with the difficulties of complex and antigenic variation of the plasmodium. The third functional domain (AMA-1 (III)) of the main merozoite surface antigen 1 (MSP1) 19kD C-terminal region (MSP1-19) and the merozoite top membrane antigen 1 of the plasmodium falciparum are the fragments of the enzyme solution when the merozoites invade red blood cells and play a role in the process of the invasion of merozoites. It's an important role, and it has become two important parts of the plasmodium's red-phase vaccine. candidate antigens. The laboratory fusion of two antigens of MSP1-19 and AMA-1 (III) through a sequence of linked regions (a polypeptide consisting of 28 amino acid residues, designated P28) to form a fusion antigen, i.e., PfCP-2.9. Fusion protein. The fusion antigen has been completed as a candidate antigen for the malaria vaccine Clinical trials. The PfCP-2. 9 recombinant malaria vaccine has shown significant advantages over foreign similar vaccines: high immunogenicity and strong inhibition of plasmodium Long efficacy. The antibody drop is increased by 22 and 47 fold, respectively, compared to the two antigen components; the fusion antigen immunogenicity enhances the ability of the immune serum to completely inhibit the growth of the parasite at a concentration of 15%, while the individual antigen is immunized or mixed with two antigens The immune response of PfCP-2. 9 fusion antigen was higher than that of single antigen. In the expression system of Pichia pastoris, the expression of PfCP-2.9 fusion antigen was 2. 6 g/ l, which is about 20 times higher than that of a single antigen in the same system, and is soluble and stable In this lab, a group of monoclonal antibodies were prepared by using the PfCP-2.9 recombinant protein in the earlier study, which was mainly used in the mechanism of the immune protection of the PfCP-2.9 vaccine, including the surface identification of the PfCP-2.9 fusion antigen, and the immune serum with the PfCP-2.9 vaccine. The competitive ELISA test is used to analyze whether some important table bits of the vaccine are effectively delivered to the human body The results of these studies will partially illustrate the mechanism of the immune protection of the PfCP-2. 9 fusion antigen vaccine and provide a vaccine for the fusion of the antigen. The basis and information of the design were provided. The 21 strains of PfCP-2.9 were fused in this study. The characteristics of the monoclonal antibody of the antigen were analyzed. A certain amount of the monoclonal antibody was prepared by the cultivation of the hybridoma cells and the intraperitoneal injection of BALB/ c mice. The following studies were carried out and the results are as follows: (1) the reactivity of the monoclonal antibody and the fusion protein: 21 strains were detected by ELISA The results were positive, and the other 3 strains were negative. The results were detected by Western blot. The results were as follows: 18 strains were positive; (2) the identification of the monoclonal antibody was carried out: the PfCP-2.9 antigen was completely denatured with urea, and then the protein was added with the modified and undenatured PfCP-2.9protein. Western blot showed that 6 of them can recognize the denatured and undenatured protein, indicating that these monoclonal antibodies recognize the linear table position, while the remaining 12 monoclonal antibodies do not recognize the modified PfCP-2.9 fusion antigen, indicating that these monoclonal antibodies recognize the conformation table. Location: (3) Analysis of the identification area of the monoclonal antibody: The MSP1-19 prepared in the laboratory and the AMA-1 standard provided by the NIH are the antigen, and the specific identification area of the 18 monoclonal antibodies is detected by Western blot. The result is that 5 strains of the monoclonal antibody of MSP1-19 are identified, 8 of the monoclonal antibodies to the AMA-1 are identified, and the remaining 5 Both MSP1-19 and AMA-1 were not identified, but the PfCP-2. 9 fusion protein was identified. (4) Table-position competitive assay: PfCP-2.9 was used as the antigen, and the antibody and HRP-labeled monoclonal antibody were used in the competitive ELISA. The results show that 4 pairs of monoclonal antibodies are competing with each other, suggesting that the 4 pairs of monoclonal antibodies may identify the same table or that the identified table bits have an interaction with the conformation or in the identification sequence; and (5) the reactivity of the monoclonal antibody against the natural protein of the plasmodium: The worm (FCC1/ HN strain) is an antigen and is used in the form of a blood smear by an indirect immunofluorescence method (IFA) The biological activity of the cloned antibody was determined. The results showed that 8 monoclonal antibodies could not The effect of (6) single-antibody on the growth of plasmodium falciparum was studied. The results showed that in the system with 2% red blood cells and 0.5% of the initial protozoa, the LDH method was used to measure the growth of plasmodium. The effect of monoclonal antibody on the growth of plasmodium was determined. The results showed that when the final concentration of the monoclonal antibody was 0.5mg/ ml, there were 6. The growth of plasmodium was inhibited by the different degree of monoclonal antibody. The recombinant eggs of PfCP-2.9were studied in this study. The results of this study provide important information and foundation for the study on the mechanism of the immune protection of the PfCP-2.9malaria vaccine
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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