恶性疟原虫PfMAg-1对裂殖子入侵功能的影响
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摘要
恶性疟原虫PfMAg-1蛋白为裂殖子表面相关蛋白,为了探究PfMAg-1的功能,本研究采用规律成簇间隔短回文重复及相关失活蛋白(Clustered regulatory interspaced short palindromic repeats/dead CRISPRassociated protein 9,CRISPR/d Cas9)技术,构建恶性疟原虫PfMAg-1低表达质粒,电穿孔方法转染后通过抗稻瘟菌素(Blasticidin S Deaminase,BSD)筛选出恶性疟原虫MAg1~(KD)(Knock Down,KD)虫株。通过聚合酶链式反应(Polymerase Chain Reaction,PCR)、实时荧光定量聚合酶链式反应(quantitative Real Time Polymerase Chain Reaction,q RT-PCR)检测PfMAg-1表达水平。在体外培养条件下观察MAg1~(KD)虫株生长趋势及裂殖子入侵效率来评价PfMAg-1的表达对恶性疟原虫红内期增殖的影响。结果表明,恶性疟原虫MAg1~(KD)虫株较对照虫株的体外生长率显著降低,进一步实验提示影响疟原虫增殖速度的原因是MAg1~(KD)虫株裂殖子入侵效率下降。本研究证实PfMAg-1在恶性疟原虫裂殖子入侵红细胞时发挥重要作用,为进一步评价PfMAg-1蛋白作为疟疾疫苗候选抗原的前景提供了实验依据。
PfMAg-1 protein of Plasmodium falciparum( P. falciparum) is a merozoite surface-associated protein. To explore the function of PfMAg-1,the technic of clustered regulatory interspaced short palindromic repeats/dead CRISPRassociated protein 9( CRISPR/dCas9) was applied to construct low expression plasmid of PfMAg-1,and then to screen the MAg1~(KD) strains by blasticidin S deaminase( BSD) after transfection by electroporation. The expression level of PfMAg-1 was detected by PCR and q RT-PCR,the effect of PfMAg-1 expression on the proliferation in intro erythrocytic stage of P.falciparum was evaluated by observing the growth trend of MAg1~(KD) strains and the efficiency of merozoite invasion in vitro.The results showed that the Pf MAg-1 expression level of MAg1~(KD) strain was significantly lower than that of the control strain,resulting in a decrease in the growth rate of the strain in vitro. Further experiments confirmed that the factor affecting the proliferation speed was the decreased efficiency of merozoite invasion of the MAg1~(KD) strain. The present study confirmed that PfMAg-1 has a significant effect on merozoite invasion,indicating that PfMAg-1 protein plays an important role in the merozoite invasion in intro erythrocytic stage of P. falciparum,so it presents future prospects of PfMAg-1 protein as a malaria vaccine candidate.
PfMAg-1 protein of Plasmodium falciparum( P. falciparum) is a merozoite surface-associated protein. To explore the function of PfMAg-1,the technic of clustered regulatory interspaced short palindromic repeats/dead CRISPRassociated protein 9( CRISPR/dCas9) was applied to construct low expression plasmid of PfMAg-1,and then to screen the MAg1~(KD) strains by blasticidin S deaminase( BSD) after transfection by electroporation. The expression level of PfMAg-1 was detected by PCR and q RT-PCR,the effect of PfMAg-1 expression on the proliferation in intro erythrocytic stage of P.falciparum was evaluated by observing the growth trend of MAg1~(KD) strains and the efficiency of merozoite invasion in vitro.The results showed that the Pf MAg-1 expression level of MAg1~(KD) strain was significantly lower than that of the control strain,resulting in a decrease in the growth rate of the strain in vitro. Further experiments confirmed that the factor affecting the proliferation speed was the decreased efficiency of merozoite invasion of the MAg1~(KD) strain. The present study confirmed that PfMAg-1 has a significant effect on merozoite invasion,indicating that PfMAg-1 protein plays an important role in the merozoite invasion in intro erythrocytic stage of P. falciparum,so it presents future prospects of PfMAg-1 protein as a malaria vaccine candidate.
引文
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Chiu CY,White MT,Healer J et al.Different regions of Plasmodium falciparum erythrocyte-binding antigen 175 induce antibody responses to infection of varied efficacy.J Infect Dis.,2016,214(1):96-104.
Crunkhorn,S.Malaria:Clues to vaccine design.Nat Rev Drug Discov.,2018,17(5):316.
Cui Y,Xu J,Cheng M,Liao X,Peng S.Review of CRISPR/Cas9sg RNA Design Tools.Interdiscip Sci.,2018,10(2):455-456.
de Koning-Ward TF,O’Donnell RA,Drew DR,Thomson R,Speed TP,Crabb BS.A new rodent model to assess blood stage immunity to the Plasmodium falciparum antigen merozoite surface protein 119 reveals a protective role for invasion inhibitory antibodies.J Exp Med.,2003,198(6):869-875.
Gao FM.Immunological function of the N-terminus of Pf MAg-1,a protective antigen of Plasmodium falciparum.Master Dissertation of Chinese Academy of Medical Science.2002,1-3.[高芳明.恶性疟原虫红内期保护性抗原Pf MAg-1N末端免疫学功能研究.中国医学科学院硕士学位论文,2002,1-3.]
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Gao YH,Li HL,Lu Y,Gao FM,Lin YH,Zhou HC,Zhang LH,Wang H.Identification of a vaccine candidate antigen,Pf MAg-1,from Plasmodium falciparum with monoclonal antibody M26-32.Parasitol Res.,2009,105(6):1 723-1 732.
Guo L.Establishment of transfection technology platform of Plasmodium falciparum.Master Dissertation of Chinese Academy of Medical Science,2015,2-3.[郭莉.恶性疟原虫转染技术平台的建立.中国医学科学院硕士学位论文,2015,2-3.]
Koch M,Wright KE,Otto O,Herbig M,Salinas ND,Tolia NH,Satchwell TJ,Guck J,Brooks NJ,Baum J.Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion.Proc Natl Acad Sci USA,2017,114(16):4 225-4 230.
Lu Y.Cloning and functional analysis of a new Plasmodium falciparum protective antigen gene Pf MAg-1.Master Dissertation of Chinese Academy of Medical Science.2001,1-2.[路妍.一种新恶性疟原虫保护性抗原基因Pf MAg-1的克隆及其功能研究.中国医学科学院硕士学位论文,2001,1-2.]
Ogutu BR,Apollo OJ,Mc Kinney D et al.Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya.PLoS One,2009,4(3):e4708.
Patarroyo ME,Alba MP,Rojas-Luna R,Bermudez A,Aza-Conde J.Functionally relevant proteins in Plasmodium falciparum host cell invasion.Immunotherapy,2017,9(2):131-155.
Paul G,Deshmukh A,Kumar Chourasia B et al.Protein-protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation tha binds to human erythrocytes.Biochem J.,2018,475(6):1197-1 209.
Pulecio J,Verma N,Mejia-Ramirez E,Huangfu D,Raya A.CRISPR/Cas9-Based Engineering of the Epigenome.Cell Stem Cell,2017,21(4):431-447.
Riley EM,Allen SJ,Wheeler JG et al.Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen(Pf MSP1)of Plasmodium falciparum are associated with reduced malaria morbidity.Parasite Immunol.,1992,14(3):321-337.
Simon JE,Rodriguez AS,Santiago VN.CRISPR-Cas9:A precise approach to genome engineering.Ther Innov Regul Sci,2018,2168479018762798.
Thera MA,Doumbo OK,Coulibaly D et al.Safety and allele-specific immunogenicity of a malaria vaccine in Malian adults:results of a phase I randomized trial.PLoS Clin Trials.,2006,1(7):e34.
Burns JM,Jr.Adeeku EK,Dunn PD.Protective immunization with a novel membrane protein of Plasmodium yoelii-infected erythrocytes.Infect Immun.,1999,67(2):675-680.
Capecchi MR.Altering the genome by homologous recombination.Science,1989,244(4910):1 288-1 292.
Chiu CY,White MT,Healer J et al.Different regions of Plasmodium falciparum erythrocyte-binding antigen 175 induce antibody responses to infection of varied efficacy.J Infect Dis.,2016,214(1):96-104.
Crunkhorn,S.Malaria:Clues to vaccine design.Nat Rev Drug Discov.,2018,17(5):316.
Cui Y,Xu J,Cheng M,Liao X,Peng S.Review of CRISPR/Cas9sg RNA Design Tools.Interdiscip Sci.,2018,10(2):455-456.
de Koning-Ward TF,O’Donnell RA,Drew DR,Thomson R,Speed TP,Crabb BS.A new rodent model to assess blood stage immunity to the Plasmodium falciparum antigen merozoite surface protein 119 reveals a protective role for invasion inhibitory antibodies.J Exp Med.,2003,198(6):869-875.
Gao FM.Immunological function of the N-terminus of Pf MAg-1,a protective antigen of Plasmodium falciparum.Master Dissertation of Chinese Academy of Medical Science.2002,1-3.[高芳明.恶性疟原虫红内期保护性抗原Pf MAg-1N末端免疫学功能研究.中国医学科学院硕士学位论文,2002,1-3.]
Gao YH.Evaluation of immunoprotective effect of Pf MAg-1 candidate antigen of Plasmodium falciparum.Doctor Dissertation of Chinese Academy of Medical Science,2007,139-140.[高宇辉.恶性疟原虫候选抗原Pf MAg-1的免疫保护效果评价.中国医学科学院博士学位论文,2007,139-140.]
Gao YH,Li HL,Lu Y,Gao FM,Lin YH,Zhou HC,Zhang LH,Wang H.Identification of a vaccine candidate antigen,Pf MAg-1,from Plasmodium falciparum with monoclonal antibody M26-32.Parasitol Res.,2009,105(6):1 723-1 732.
Guo L.Establishment of transfection technology platform of Plasmodium falciparum.Master Dissertation of Chinese Academy of Medical Science,2015,2-3.[郭莉.恶性疟原虫转染技术平台的建立.中国医学科学院硕士学位论文,2015,2-3.]
Koch M,Wright KE,Otto O,Herbig M,Salinas ND,Tolia NH,Satchwell TJ,Guck J,Brooks NJ,Baum J.Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion.Proc Natl Acad Sci USA,2017,114(16):4 225-4 230.
Lu Y.Cloning and functional analysis of a new Plasmodium falciparum protective antigen gene Pf MAg-1.Master Dissertation of Chinese Academy of Medical Science.2001,1-2.[路妍.一种新恶性疟原虫保护性抗原基因Pf MAg-1的克隆及其功能研究.中国医学科学院硕士学位论文,2001,1-2.]
Ogutu BR,Apollo OJ,Mc Kinney D et al.Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya.PLoS One,2009,4(3):e4708.
Patarroyo ME,Alba MP,Rojas-Luna R,Bermudez A,Aza-Conde J.Functionally relevant proteins in Plasmodium falciparum host cell invasion.Immunotherapy,2017,9(2):131-155.
Paul G,Deshmukh A,Kumar Chourasia B et al.Protein-protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation tha binds to human erythrocytes.Biochem J.,2018,475(6):1197-1 209.
Pulecio J,Verma N,Mejia-Ramirez E,Huangfu D,Raya A.CRISPR/Cas9-Based Engineering of the Epigenome.Cell Stem Cell,2017,21(4):431-447.
Riley EM,Allen SJ,Wheeler JG et al.Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen(Pf MSP1)of Plasmodium falciparum are associated with reduced malaria morbidity.Parasite Immunol.,1992,14(3):321-337.
Simon JE,Rodriguez AS,Santiago VN.CRISPR-Cas9:A precise approach to genome engineering.Ther Innov Regul Sci,2018,2168479018762798.
Thera MA,Doumbo OK,Coulibaly D et al.Safety and allele-specific immunogenicity of a malaria vaccine in Malian adults:results of a phase I randomized trial.PLoS Clin Trials.,2006,1(7):e34.