惡性瘧原蟲Pf332抗原的免疫原性分析
發(fā)布時(shí)間：2019-04-21 17:47
【摘要】： 瘧原蟲抗藥性的產(chǎn)生和擴(kuò)散已使全球瘧疾疫情嚴(yán)重惡化,每年瘧疾死亡人數(shù)自上世紀(jì)七十年代的約50萬上升至目前約100萬,藥物防治瘧疾的措施亦面臨嚴(yán)重的困難和挑戰(zhàn)。因此新的瘧疾控制措施的實(shí)施已成為當(dāng)務(wù)之急。疫苗接種已使許多種傳染病得到有效的控制乃至根除,因此研制有效的瘧疾疫苗成為防止瘧原蟲感染的重要手段。 Pf332是惡性瘧原蟲基因組所編碼的分子量最大的蛋白質(zhì)。該蛋白質(zhì)具有典型的跨膜蛋白結(jié)構(gòu),其膜外區(qū)含有一個(gè)保守的Duffy抗原結(jié)合基團(tuán)相類似的功能區(qū)(Duffy-binding like, DBL domain)。Pf332由紅內(nèi)期惡性瘧原蟲分泌運(yùn)輸?shù)礁腥镜募t細(xì)胞表面,在裂殖子侵入紅細(xì)胞的過程中起到了輔助作用。由于其膜外DBL區(qū)與其他膜外區(qū)蛋白質(zhì)相比具有高度的保守性,使其可能成為發(fā)展新的抗瘧藥物和疫苗的候選分子。本研究制備得到12株抗Pf332-DBL區(qū)重組蛋白質(zhì)的IgG1亞型單克隆抗體,通過Western blot和間接免疫熒光實(shí)驗(yàn)驗(yàn)證了這12株單抗不僅可以特異性識(shí)別重組蛋白質(zhì),而且可以識(shí)別蟲體天然蛋白Pf332。在此基礎(chǔ)上,根據(jù)Pf332-DBL區(qū)氨基酸序列合成9段多肽,以肽掃描的方法確定了單抗的結(jié)合肽段,通過單抗體外抑制蟲體侵入實(shí)驗(yàn),發(fā)現(xiàn)Pf332-DBL區(qū)在瘧原蟲裂殖子侵入紅細(xì)胞過程中發(fā)揮重要作用的兩個(gè)功能區(qū)域位于第1~25個(gè)氨基酸和第76~100個(gè)氨基酸。在分析Pf332-DBL區(qū)重要生物學(xué)功能的同時(shí),為確定其在瘧疾疫苗研究中的可應(yīng)用性,本研究還對(duì)Pf332-DBL區(qū)重組蛋白質(zhì)的免疫原性進(jìn)行了分析。通過比較Pf332-DBL區(qū)重組蛋白質(zhì)與4種不同佐劑混合免疫動(dòng)物后的抗體效價(jià)、抗體亞型及抗體維持水平,揭示出Pf332-DBL區(qū)具有很強(qiáng)的免疫原性,而且篩選到適合Pf332-DBL區(qū)在疫苗研究中的佐劑ISA720。 本研究發(fā)現(xiàn)Pf332是瘧原蟲的一個(gè)重要功能蛋白質(zhì),DBL區(qū)是Pf332蛋白實(shí)施生物學(xué)功能的關(guān)鍵區(qū)域。Pf332-DBL區(qū)具有很強(qiáng)的免疫原性,是抗瘧原蟲的重要疫苗候選抗原。
[Abstract]:The emergence and spread of drug resistance of Plasmodium malaria has seriously worsened the global malaria epidemic. The number of malaria deaths per year has increased from about five hundred thousand in the 1970s to about 1 million at present. The measures to control malaria by drugs are also facing serious difficulties and challenges. Therefore, the implementation of new malaria control measures has become a top priority. Vaccination has resulted in the effective control and eradication of many infectious diseases. Therefore, the development of effective malaria vaccines has become an important means to prevent the infection of Plasmodium falciparum. Pf332 is the largest protein encoded by Plasmodium falciparum genome. The protein has a typical transmembrane protein structure, and its extracellular region contains a conserved Duffy antigen-binding group-like functional area (Duffy-binding like, DBL domain). Pf332) secreted by Plasmodium falciparum in the erythrocytic stage to transport to the surface of infected red blood cells. It plays an auxiliary role in the process of merozoite invading red blood cells. Because of its highly conservative nature compared with other proteins in the extracellular DBL region, it may become a candidate molecule for the development of new antimalarial drugs and vaccines. In this study, 12 monoclonal antibodies against Pf332-DBL domain recombinant protein IgG1 subtype were prepared. The results of Western blot and indirect immunofluorescence test showed that these 12 monoclonal antibodies not only specifically recognized the recombinant protein, but also showed that these monoclonal antibodies could specifically recognize the recombinant protein. And it can recognize the natural protein Pf332.. On the basis of this, nine peptides were synthesized according to the amino acid sequence of Pf332-DBL region. The binding peptides of monoclonal antibodies were determined by peptide scanning, and the in vitro inhibition of insect invasion by monoclonal antibodies was carried out. It was found that the two functional regions which play an important role in the invasion of erythrocytes by merozoites of Plasmodium falciparum are located at 1 ~ 25 amino acids and 76 ~ 100 amino acids. In order to determine the applicability of Pf332-DBL region in malaria vaccine research, the immunogenicity of recombinant protein in Pf332-DBL region was also analyzed in this study. By comparing the antibody titer, antibody subtype and antibody maintenance level between the recombinant protein of Pf332-DBL region and four different adjuvants, it was revealed that the Pf332-DBL region had a strong immunogenicity. And the adjuvant ISA720., which is suitable for Pf332-DBL region in vaccine research, was screened out. In this study, we found that Pf332 is an important functional protein of Plasmodium malaria, and DBL region is the key region of the biological function of Pf332 protein. Pf332-DBL region has strong immunogenicity and is an important vaccine candidate antigen of Plasmodium malaria.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：2462421
[Abstract]:The emergence and spread of drug resistance of Plasmodium malaria has seriously worsened the global malaria epidemic. The number of malaria deaths per year has increased from about five hundred thousand in the 1970s to about 1 million at present. The measures to control malaria by drugs are also facing serious difficulties and challenges. Therefore, the implementation of new malaria control measures has become a top priority. Vaccination has resulted in the effective control and eradication of many infectious diseases. Therefore, the development of effective malaria vaccines has become an important means to prevent the infection of Plasmodium falciparum. Pf332 is the largest protein encoded by Plasmodium falciparum genome. The protein has a typical transmembrane protein structure, and its extracellular region contains a conserved Duffy antigen-binding group-like functional area (Duffy-binding like, DBL domain). Pf332) secreted by Plasmodium falciparum in the erythrocytic stage to transport to the surface of infected red blood cells. It plays an auxiliary role in the process of merozoite invading red blood cells. Because of its highly conservative nature compared with other proteins in the extracellular DBL region, it may become a candidate molecule for the development of new antimalarial drugs and vaccines. In this study, 12 monoclonal antibodies against Pf332-DBL domain recombinant protein IgG1 subtype were prepared. The results of Western blot and indirect immunofluorescence test showed that these 12 monoclonal antibodies not only specifically recognized the recombinant protein, but also showed that these monoclonal antibodies could specifically recognize the recombinant protein. And it can recognize the natural protein Pf332.. On the basis of this, nine peptides were synthesized according to the amino acid sequence of Pf332-DBL region. The binding peptides of monoclonal antibodies were determined by peptide scanning, and the in vitro inhibition of insect invasion by monoclonal antibodies was carried out. It was found that the two functional regions which play an important role in the invasion of erythrocytes by merozoites of Plasmodium falciparum are located at 1 ~ 25 amino acids and 76 ~ 100 amino acids. In order to determine the applicability of Pf332-DBL region in malaria vaccine research, the immunogenicity of recombinant protein in Pf332-DBL region was also analyzed in this study. By comparing the antibody titer, antibody subtype and antibody maintenance level between the recombinant protein of Pf332-DBL region and four different adjuvants, it was revealed that the Pf332-DBL region had a strong immunogenicity. And the adjuvant ISA720., which is suitable for Pf332-DBL region in vaccine research, was screened out. In this study, we found that Pf332 is an important functional protein of Plasmodium malaria, and DBL region is the key region of the biological function of Pf332 protein. Pf332-DBL region has strong immunogenicity and is an important vaccine candidate antigen of Plasmodium malaria.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 文雪霞;陳化蘭;熊永忠;王秀榮;;抗原表位鑒定方法的研究進(jìn)展[J];中國(guó)畜牧獸醫(yī);2012年07期
相關(guān)碩士學(xué)位論文 前1條
1 武闖;日本血吸蟲23kDa膜蛋白與人免疫球蛋白的相互作用分析[D];吉林大學(xué);2011年
，本文編號(hào)：2462421
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2462421.html
最近更新
教材專著
