重組瘧疾疫苗PfCP-2.9單克隆抗體的特性分析
發(fā)布時間：2018-11-20 05:44
【摘要】： 瘧疾是世界上分布和流行最廣、危害最為嚴重的寄生蟲性傳染病之一,世界上有20億人口生活在疫區(qū)。由于瘧原蟲抗藥性以及蚊媒抗殺蟲劑抗性的產生和擴散,這給瘧疾防治帶來了很大的困難。盡管瘧疾疫苗研制也面臨瘧原蟲生活史復雜和抗原變異等困難,但研制有效的瘧疾疫苗仍是人們期望能用于控制瘧疾的潛在新途徑。 惡性瘧原蟲主要裂殖子表面抗原1(MSP1)19kD C-末端區(qū)域(MSP1-19)與裂殖子頂端膜抗原1的第三功能域(AMA-1(III))均是裂殖子入侵紅細胞時被酶解的片段,在裂殖子入侵過程中發(fā)揮重要作用,已成為瘧原蟲紅內期疫苗的兩個重要候選抗原。本實驗室將MSP1-19和AMA-1(III)兩個抗原通過一段鏈接區(qū)序列(由28個氨基酸殘基組成的多肽,定名為P28)進行融合,形成融合抗原,即PfCP-2.9融合蛋白。該融合抗原作為瘧疾疫苗候選抗原已完成2個臨床試驗。與國外同類疫苗相比,此種PfCP-2.9重組瘧疾疫苗已顯示出明顯的優(yōu)點:①高免疫原性和強抑制瘧原蟲生長的效力。與兩個抗原成分相比,抗體滴度分別增加22和47倍;融合抗原免疫原性增強使其免疫血清在15%濃度時能完全抑制瘧原蟲生長,而單個抗原免疫或將兩個抗原混合后免疫,其免疫血清在15%濃度時對瘧原蟲生長無明顯抑制作用;②不同地區(qū)瘧疾病人血清對PfCP-2.9融合抗原的免疫反應性比單抗原成分明顯增強;③在畢氏酵母分泌表達系統中,PfCP-2.9融合抗原表達產量達到2.6克/升,比單抗原在同一系統中表達產量高約20倍,且可溶性和穩(wěn)定性明顯提高。 本實驗室在前期研究中采用PfCP-2.9重組蛋白制備了一組單克隆抗體,主要用于PfCP-2.9疫苗免疫保護作用的機制探討,包括PfCP-2.9融合抗原的表位鑒定,通過與PfCP-2.9疫苗接種者免疫血清的競爭ELISA試驗,分析該疫苗的一些重要表位在人體是否得到有效遞呈,產生免疫應答等。這些研究預期結果將部分闡明PfCP-2.9融合抗原疫苗的免疫保護作用機制,并為融合抗原疫苗的設計提供依據和信息。 本研究對21株PfCP-2.9融合抗原單克隆抗體的特性進行分析。通過雜交瘤細胞的培養(yǎng)和BALB/c小鼠腹腔注射制備一定量的單克隆抗體,在此基礎上開展以下的研究并取得結果如下:(1)單抗與融合蛋白的反應性:用ELISA檢測21株單抗,結果為陽性反應的18株,另外3株為陰性;用Western blot方法檢測結果:同樣為18株陽性;(2)單克隆抗體識別表位構象分析:將PfCP-2.9抗原用尿素進行徹底變性,再用單抗與變性和未變性PfCP-2.9蛋白進行Western blot反應,結果顯示其中6株可以識別變性和未變性蛋白,表明這些單抗識別的是線性表位;而剩下的12株單抗不識別變性后的PfCP-2.9融合抗原,表明這些單抗識別的是構象表位;(3)單抗識別區(qū)域的分析:以本實驗室制備的MSP1-19和美國NIH提供的AMA-1標準品為抗原,用Western blot的方法對18株單抗的特異性識別區(qū)域進行檢測,結果為識別MSP1-19的單抗共5株,而識別AMA-1的單抗有8株,剩余的5株單抗既不識別MSP1-19,也不識別AMA-1,但識別PfCP-2.9融合蛋白;(4)表位競爭性分析:以PfCP-2.9為抗原,用競爭ELISA方法先后用過量未標記單抗和HRP標記單抗與之反應,結果有4對單抗出現相互競爭,提示這4對單抗可能識別同一表位或者是識別的表位在構象上或者在識別序列上存在相互影響;(5)單克隆抗體與瘧原蟲天然蛋白反應性:以處于成熟裂殖體期的惡性瘧原蟲(FCC1/HN株)為抗原,以血涂片的形式用間接免疫熒光方法(IFA)對單克隆抗體的生物學活性進行測定。結果顯示:有8株單抗能夠不同程度的與瘧原蟲天然蟲體反應。(6)單抗體外抑制瘧原蟲生長的效力分析:在紅細胞壓積到2%,起始原蟲率為0.5%的體系中,采用LDH方法分別測定各單抗抑制瘧原蟲生長的效力。結果表明:當單抗終濃度為0.5mg/ml時,有6株單抗不同程度抑制瘧原蟲生長。 本研究對PfCP-2.9重組蛋白的一組單克隆抗體的特性進行了初步分析。本研究的結果為開展PfCP-2.9瘧疾疫苗免疫保護作用機制研究提供重要信息和基礎,繼而為設計和完善融合抗原瘧疾疫苗提供依據。
[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
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：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
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
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