Construction And Identification Of Genetically Modified Strain Of Plasmodium Falciparum By CRISPR/Cas9 Technique And Preliminary Study On Infection In Tree Shrew(Tupaia Belangeri)

Falciparum malaria is an infectious disease that is a serious threat to human health.Due to the lack of effective gene editing means and appropriate animal models,the research on the pathogenesis of malaria parasite,malaria vaccine,drug development and the study of drug-resistant strain have been slow.The analysis of plasmodium resistance from a genetic level can accelerate the study of antimalarial vaccines and drugs,as well as a hotspot and focus of plasmodium falciparum.The genome editing of plasmodium falciparum is limited by tools and low efficiency of transfection and integration.CRISPR/Cas9 technology can change the gene of plasmodium falciparum quickly,economically,efficiently and specifically,and accelerate the identification of new drug targets and the elucidation of specific genes or gene family functions,and supplement the genome-wide research,promote the development of plasmodium vaccine and new antimalarial drugs.Both casein kinase ?(CK2)and serine/threonine protein kinases(STK)are protein kinases.CK2 plays an important role in cell growth,proliferation,apoptosis and carcinogenesis.CK2 is a bidirectional specific kinase,the CK2?1 and CK2a subunit protein can be used as a candidate target for malaria drug therapy.STK is closely related to cell growth,division,metabolism,differentiation,virulence and pathogenicity.STK is also an important antigen,which can be used to develop a new malaria vaccine.STK protein and CK2 protein may be related to the physiological function and pathogenicity of plasmodium falciparum.In this study,CK2?1/CK2?/STK protein were selected for tagging gene modification,and mutation sites were introduced to construct the recombinant insect strain,and a preliminary experiment of tree shrew infection was carried out.In the absence of a corresponding commercial antibody against plasmodium protein,the specific protein of the plasmodium falciparum is screened and the gene targeted mutation is edited by using the label.It provides a basis for further study of gene function and provides a technical means for gene editing of other related proteins of plasmodium falciparum.Percoll synchronous culture method was used to obtain the circoid stage of P.falciparum 3D7.The homologous arm of CK2?1/CK2?/STK gene was amplified from genomic DNA of P.falciparum 3D7,and the HA/HA-Ty1/HA-Ty1 tag was obtained by PCR amplification without template.The homologous arm of CK2?1/CK2?/STK gene was fused with HA/HA-Ty1/HA-Ty1 tag by bypass grafting,At the same time,the nucleic acid sequence of synonymous mutation was introduced,the HA-CK2?1/HA-Ty1-CK2a/HA-Ty1-STK mutant gene were obtained.PL6CS-hDHFR plasmid was successfully constructed by using PL6CS plasmid as vector.The HA-CK2?1/HA-Ty1-CK2?/HA-Ty1-STK mutant gene was inserted into PL6CS-hDHFR plasmids,and the fusion plasmid of PL6CS-hDHFR-CK2?1/CK2?/STK was successfully constructed.The sgRNA sequence of CK2?1/CK2?/STK gene was inserted into the corresponding PL6CS-hDHFR-CK2?1/CK2?/STK fusion plasmid,PL6CS-hDHFR-CK2?1/CK2?/STK integration plasmid was successfully obtained.The Pufl-BSD-Cas9 plasmid and PL6CS-hDHFR-CK2?1/CK2?/STK integrated plasmid were transfected into plasmodium falciparum 3D7 strain by electroporation.The recombinant PL6CS-hDHFR CK2?1/CK2?/STK gene strain was successfully obtained by PCR identification,DNA sequencing,Western blotting identification and cellular immunofluorescence analysis(IFA).The vitro and vivo infection of tree shrews was studied with plasmodium falciparum gene recombinant strain.The results showed that CRISPR/Cas9 technique could be used to construct plasmodium falciparum transgenic strain successfully and quickly.The recombinant strain of PL6CS-hDHFR-CK2?1/CK2?/STK gene was cultured in serum or plasma of tree shrew in vitro,and the recombinant strain which could infect red blood cells of tree shrew in vitro was successfully obtained.CK2?1/CK2?/STK gene recombinant strains were used to infect uncut and splenic resected tree shrews in vivo.The results showed that tree shrews were not infected by three genetically modified strains.This study developed and improved the method of rapid genomic transformation of plasmodium falciparum using CRISPR/Cas9 method,which laid a foundation for the further study of plasmodium falciparum at gene level.The recombinant strain of plasmodium falciparum was successfully cultured in the erythrocytes of tree shrews in vitro.It can be used as a reference for in vitro culture of other human malaria parasites and animal malaria parasites.Although the results of the final in vivo experiments show that plasmodium falciparum can not infect tree shrews,it is also an attempt and supplement for the study of the animal model of plasmodium falciparum.