重组卵形疟原虫裂殖子表面蛋白1 N端的抗原性及免疫原性分析  

Antigenicity and immunogenicity analysis of the recombinant merozoite surface protein 1 N-terminal of Plasmodium ovale

作　　者：于嘉利 刘蕾 杨博 楚瑞林 孙毅凡 刘耀宝[4] 程洋 YU Jia-li;LIU Lei;YANG Bo;CHU Rui-lin;SUN Yi-fan;LIU Yao-bao;CHENG Yang(Laboratory of Pathogen Infection and Immunity,Wuxi School of Medicine,Jiangnan University,Wuxi 214000,China;The Third People’s Hospital of Jiangyin City,Wuxi 214000,China;Shanghai Center for Disease Control and Prevention,Shanghai 200336,China;Jiangsu Institute of Parasite Diseases,Wuxi 214000,China)

机构地区：[1]江南大学无锡医学院病原感染与免疫研究室,无锡214000 [2]江阴市第三人民医院,无锡214000 [3]上海市疾病预防控制中心,上海200336 [4]江苏省血吸虫病防治研究所,无锡214000

出　　处：《中国寄生虫学与寄生虫病杂志》2021年第6期746-752,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(81871681)。

摘　　要：目的表达卵形疟原虫柯氏亚种(Plasmodium ovale curtisi)和沃氏亚种(P.ovale wallikeri)裂殖子表面蛋白1(merozoite surface protein 1,MSP1)N端重组蛋白,并进行抗原性及免疫原性分析,以探究其作为卵形疟候选疫苗的潜力。方法PCR扩增卵形疟原虫基因组DNA Pomsp1 N端基因。将纯化后的Pocmsp1 N端、Powmsp1 N端分别与pET30a、pET32a载体连接,并转化至DH5α感受态细胞中,经Bam HⅠ和XhoⅠ双酶切验证且测序无误后,转入大肠埃希菌BL21(DE3)pLysS中。0.1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后纯化蛋白,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)检测蛋白表达情况。15只BALB/c小鼠随机分为r Poc MSP1 N端组、r Pow MSP1 N端组和阴性对照组,每组5只,分别腹腔注射50μg与弗氏完全佐剂混合的r Poc MSP1和r Pow MSP1 N端蛋白和等量的PBS,初次免疫后第21天和第42天使用弗氏不完全佐剂加强免疫,初次免疫后第0、7、28和49天采集小鼠尾静脉血,制备血清。ELISA、Western blotting检测小鼠血清中特异性IgG、分析抗体滴度及抗体亲和指数,评估纯化后的r Poc MSP1和r Pow MSP1 N端蛋白的免疫原性。Western blotting检测r Poc MSP1和r Pow MSP1 N端蛋白的交叉反应,并用蛋白芯片分析重组蛋白在卵形疟原虫感染者血清中的抗原反应性。采用GraphPad Prism 5.0软件和Microsoft Excel 2016软件进行统计学分析,组间差异比较采用成组t检验。结果PCR扩增结果显示,Pocmsp1 N端和Powmsp1 N端片段大小均为1068 bp,与预期大小一致。PCR产物经克隆、诱导并纯化后,所获r Poc MSP1和r Pow MSP1 N端蛋白浓度分别为0.5 mg/ml和1.0 mg/ml。SDS-PAGE结果显示,r Poc MSP1和r Pow MSP1 N端的相对分子质量(Mr)分别约为46000和59000,Western blotting结果证实蛋白成功表达。免疫组小鼠血清均可特异性识别相应抗原。ELISA结果显示,在免疫后第7天检测到特异性IgG抗体,纯�Objective This study aims to express the merozoite surface protein 1 N-terminal proteins of Plasmodium ovale curtisi and P.ovale wallikeri,and analyze their antigenicity and immunogenicity,revealing the potential for being as vaccine candidates of P.ovale.Methods The N-terminal of the Pomsp1 gene was amplified from the genomic DNA of P.ovale.The purified N-terminal sequences of Pocmsp1 and Powmsp1 were separately connected to the plasmid pET30a and pET32a and then transfected into DH5αcompetent cells.After verification with Bam HⅠand XhoⅠenzyme digestion and DNA sequencing,the gene sequences were transferred into Escherichia coli BL21(DE3)pLysS.After induction with 0.1 mmol/L IPTG,the expressed proteins were collected,purified,and verified with SDS-PAGE and Western blotting.Fifteen BALB/c mice were randomly assigned into three groups of 5 each:the r Poc MSP1 N-terminal protein group,rPow MSP1 N-terminal protein group,and a negative control group.The mice of the three groups were injected intraperitoneally with the recombinant protein of rPoc MSP1N-terminal and r Pow MSP1 N-terminal protein(50μg of recombinant protein emulsified with Freund’s complete adjuvant)respectively,while the control group mice were injected with PBS.On 21d and 42d after primary immunization,the mice were boosted with the corresponding antigens emulsified with Freund’s incomplete adjuvant.Furthermore,on days 0,7,28,and 49 after immunization,tail vein blood samples were collected for serum preparation.ELISA and Western blotting were used to determine the serum specific IgG,analyze antibody titer and antibody affinity index,thereby to evaluate the immunogenicity of r Poc MSP1 N-terminal and r PowMSP1 N-terminal proteins.Western blotting was used to determine the cross-reaction of the two recombinant proteins,and protein array was used to analyze their antigenicity in the serum of examinees infected with P.ovale.Results PCR demonstrated that the sequence length of Poc MSP1 N-terminal and Pow MSP1 N-terminal fragment was 1068 bp,showi

关 键 词：卵形疟原虫 裂殖子表面蛋白1 免疫原性 抗原性 

分 类 号：R382.31[医药卫生—医学寄生虫学]