伯氏疟原虫Pbg63蛋白的表达及其传播阻断活性评估  

Expression characteristics and evaluation of transmission blocking activity of candidate antigen Pbg63 in Plasmodiumberghei

作　　者：于鑫鑫 姚桂香 张迪[1] 张碧莹 刘飞[1] 杨帆[1] 闵慧 曹雅明[1] YU Xinxin;YAO Guixiang;ZHANG Di;ZHANG Biying;LIU Fei;YANG Fan;MIN Hui;CAO Yaming(College of Basic Medical Sciences,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学基础医学院,辽宁沈阳110122

出　　处：《中国病原生物学杂志》2023年第9期1047-1052,共6页Journal of Pathogen Biology

摘　　要：目的研究Pbg63蛋白在伯氏疟原虫各期的表达情况和定位,评估其作为疟疾传播阻断疫苗候选抗原的潜在效应。方法从NCBI数据库获得Pbg63基因同源序列,并进行同源序列比对;通过SMART在线网站预测其编码蛋白的结构域,利用Immunomedicine Group工具预测蛋白抗原决定簇。利用双交叉同源重组的方法在Pbg63基因C端加入HA标签,在获得阳性克隆虫株的基础上通过Western blot和间接免疫荧光试验检测该蛋白在虫体各期的表达特点和定位。用rPbg63蛋白免疫小鼠,通过酶联免疫吸附试验检测小鼠血清特异性抗体滴度,通过体外试验观察Pbg63抗血清对伯氏疟原虫雄配子体出丝中心和动合子形成的影响。结果BLAST同源序列比对显示Pbg63蛋白在不同疟原虫种属中较为保守。成功表达rPbg63蛋白,免疫小鼠后用ELISA检测血清中抗Pbg63抗体滴度为1∶25600,表明该蛋白具有良好免疫原性。Western blot检测Pbg63在疟原虫裂殖体、配子体和动合子阶段均有表达;IFA检测Pbg63在雌、雄配子体的胞浆和细胞膜均有表达,但在动合子主要表达在胞膜上。体外传播阻断试验表明,抗Pbg63免疫血清对配子体出丝中心数以及动合子的形成与对照组相比无显著影响。结论Pbg63在疟原虫各种属中相对保守,在无性期和有性期均有表达,但抗Pbg63免疫血清无明显传播阻断作用,表明Pbg63尚不能作为传播阻断疫苗候选抗原。Objective Evaluation of the expression and localization of Pbg63 protein in various stages of Plasmodium berghei and evaluation of its potential effect as a malaria transmission-blocking vaccine candidate antigen.Methods Homologous sequences of Pbg 63 in different Plasmodium species were obtained from NCBI BLAST database,the domain was predicted using SMART online site,the protein antigenic determinants were predicted by Immunomedicine Group.Double crossover homologous recombination was used to achieve the C terminal HA-tag of Pbg63,positive strains obtained by drug screening with pyrimethamine.Characterization and localization of the protein expression in plasmodium berghei stages by Western blot and indirect immunofluorescence assay.Subcutaneous immunization of mice with rPbg63 protein three times,serum specific antibody titers of Pbg63 were determined by Enzyme linked immunosorbent assay.The effect of Pbg63 antiserum on the formation of exflagellation and ookinete was determined in vitro transmission blocking assay,the serum dilution ratio is 1:5,1:10.Results The alignment results showed that the Pbg63 sequence was relatively conservative among different Plasmodium species.In this study,we successfully induced rPbg63 protein expression and the antibody titer of anti-rPbg63 sera was screened around 1:25600,indicates that the protein has strong immunogenicity.The results of pbg63 localization by Western blot indicate that Pbg63 was expressed in schizont,gametocyte and ookinete stages.Further,the localization of Pbg63 was detected by IFA assay showed that Pbg63 was located on the cytoplasm and membrane of gametocyte,while it was mainly expressed in the membrane in ookinete stage.Compare to the control group,there was no significant effect on gametocyte exflagellation(t=0.3508,P>0.05)and ookinete formation(t=0.3608,P>0.05)when the parasites cultured with Pbg63 antiserum.Conclusion Pbg63 is a relatively conserved protein in Plasmodium and highly expressed in both asexual and sexual relatively conserved protein in P

关 键 词：疟疾 伯氏疟原虫 传播阻断疫苗 Pbg63蛋白 

分 类 号：R382.31[医药卫生—医学寄生虫学]