传播阻断疫苗候选抗原Pb280在伯氏疟原虫中的表达及功能研究

Expression and function of a potential antigen Pb280 as transmission-blocking vaccine in Plasmodium berghei

目的探讨传播阻断疫苗候选抗原Pb280(PBANKA_041710)在伯氏疟原虫中的表达情况及基因功能。方法从NCBI数据库获取不同疟原虫种属Pb280氨基酸同源序列,用MEGA7构建系统进化树。用SMART在线网站预测Pb280蛋白结构域。昆明小鼠经尾静脉注射1×10^6个Pb感染红细胞(iRBC),当小鼠原虫血症达3%~5%时取眼球血进行体外培养并分离裂殖体。将带血凝素(HA)的Pb280标签质粒(Pb280HA)和Pb280敲除质粒(Pb280KO)线性化后分别转染裂殖体,用转染后裂殖体注射感染小鼠(1×10^7个裂殖体/鼠),PCR鉴定感染小鼠中的转基因疟原虫,对转基因疟原虫进行单克隆筛选,获得1株Pb280HA标签虫株和2株Pb280KO虫株(分别命名为Pb280HA型、Pb280KO-C1型、Pb280KO-C2型虫株)。蛋白质免疫印迹分析(Western blotting)和间接免疫荧光试验(IFA)分析Pb280在伯氏疟原虫中的表达情况。取9只雌性昆明小鼠随机分为基因敲除C1组(C1组)、基因敲除C2组(C2组)和对照组,每组3只,每鼠分别经尾静脉注射5×10^6个Pb280KO-C1型、Pb280KO-C2型和野生型(WT)伯氏疟原虫。感染后第3~10天,每天取小鼠尾静脉血进行血涂片检测,计算原虫血症;计算感染后第3天雌雄配子体比及配子体率。感染后第3天取小鼠尾静脉血与动合子培养基混合培养15 min后计数雄配子体出丝中心数、雌雄配子结合数、雌配子数,继续培养24 h后计数动合子数。原虫血症、配子体率和雌雄配子体比的比较采用χ^2检验,其余各组数据之间的比较采用单因素方差分析。结果系统进化树显示,Pb280与约氏疟原虫PY04819的亲缘关系最近。SMART预测结果显示,Pb280含有一个N端信号肽、10个跨膜区和1个生长因子受体结构域。Western blotting分析显示,Pb280蛋白在伯氏疟原虫中有表达,相对分子质量(Mr)约280000。IFA检测结果显示,在配子体到动合子的发育过程中,Pb280从胞浆向质膜表面分泌。基因功能分析结果显示,感染后第3天,3组小鼠原虫血症均约为15%,感染后第10天均达60%以上,C1组、C2组与对照组的差异无统计学意义(P>0.05)。感染后第3天,C1组配子体率、雌雄配子体比、平均每10个视野配子体出丝中心数量、雌雄配子结合数和雌配子数分别为39.50‰、1.65、(21.63±4.03)个、(12.50±8.02)个和(930.00±79.20)个,C2组分别为34.50‰、1.71、(18.25±5.85)个、(13.75±9.54)个和(885.00±130.11)个,对照组分别为41.50‰、1.74、(21.44±4.73)个、(15.31±8.06)个和(1018.50±58.69)个,3组比较差异均无统计学意义(P>0.05)。C1组动合子形成数量为(410.00±67.88)个,C2组为(557.50±2.12)个,对照组为(782.00±41.01)个,C1和C2组动合子数量均少于对照组(P<0.05),C1组和C2组差异无统计学意义(P>0.05)。结论Pb280在疟原虫种属中保守,在伯氏疟原虫的裂殖体、配子体及动合子阶段均有表达,敲除该基因可导致动合子数量减少。

Objective To investigate the expression and function of a transmission-blocking vaccine candidate Pb280(PBANKA_041710)in Plasmodium berghei.Methods Homologous sequences of Plasmodium species were downloaded from NCBI and compared using MEGA7.The domains of Pb280 were predicted by SMART.Ten female Kunming(KM)mice aged 6-8 weeks were randomly selected,injected with 1×10^6 P.berghei-infected red blood cells(iRBC)through tail vein.When the parasitemia in the mice reached 3%-5%,orbital blood was obtained and cultured in vitro for isolation of schizonts.The hemagglutinin(HA)tagged plasmid(Pb280HA)and Pb280 knockout plasmid(Pb280KO)were linearized and transfected to mature schizonts,which were then injected in the mice(1×10^7 schizonts/mouse)through tail vein;transgenic parasites in the infected mice was identified by PCR,and underwent monoclonal screening.As a result,one strain of Pb280HA and two strains of Pb280KO(Pb280KO-C1 and Pb280KO-C2)were obtained.The expression of Pb280 in P.berghei was analyzed by Western blotting and indirect immunofluorescence assay(IFA).Another nine female KM mice were randomly divided into 3 groups(n=3):Pb280KO-C1 group(C1 group),Pb280KO-C2 group(C2 group)and control group.Each mouse was injected with 5×10^6 Pb280KO-C1,Pb280KO-C2 or wild-type(WT)P.berghei through tail vein.From day 3 to day 10 post-infection,mice tail vein blood was sampled for monitoring daily parasitemia by examining blood smears.On day 3 after infection,gametocytemia and female/male gametes ratio were calculated;mice tail vein blood was mixed with ookinete culture medium for 15 min to count the number of exflagellation centers,number of male-female integrating and number of macrogametes followed by further culture for 24 h to count the number of ookinetes.The comparisons of parasitemia,gametocytemia and female/male gamete ratio were performed by Chi-square test,and other data were analyzed with one-way ANOVA.Results Phylogenetic tree constructed by MEGA7 showed that Pb280 was genetically most close to the PY04819 of P.yoelii.SMART prediction showed that Pb280 contained an N-terminal signal peptide,10 transmembrane domains and 1 growth factor receptor domain.Western blotting demonstrated that Pb280 protein expression occurred in P.berghei,with a relative molecular weight of 280000.IFA results showed that Pb280 was secreted from cytoplasm to plasma membrane during the development from gametocyte to ookinete.Gene function analysis revealed that on day 3 after infection,the three groups had parasitemia of about 15%,which reached over 60%on day 10 post-infection.The parasitemia rate in the group C1 and the C2 did not differ significantly from that in the control group(P>0.05).On day 3 post-infection,the gametocytemia rate,gamete sex ratio,number of exflagellation centers per 10 fields of view,number of male-female integrating and number of macrogametes in group C1 were 39.50‰,1.65,21.63±4.03,12.50±8.02,and 930.00±79.20,respetively;those in the group C2 were 34.50‰,1.71,18.25±5.85,13.75±9.54,and 885.00±130.11,respetively;and those in the control group were 41.50‰,1.74,21.44±4.73,15.31±8.06,and 1018.50±58.69.There were no statistically significant differences among the three groups(P>0.05).The numbers of ookinetes in the C1,C2 and control groups were 410.00±67.88,557.50±2.12 and 782.00±41.01,respectively.The numbers of ookinetes in the C1 and C2 groups did not differ significantly,but both were lower than that in the control group(P<0.05).Conclusion Pb280 is genetically conserved in Plasmodium spp.and expressed in schizont,gametocyte and ookinete stages of P.berghei.Knockout Pb280 may result in the decrease of ookinete formation.