Expression And Antigenicity Analysis Of Babesia Microti Truncated Secreted Antigen 1(Tbmsal) And Establishment Of Double Antigen Sandwish ELISA Based On The Recombinant Protein

Babesiosis is a zoonotic parasitosis,caused by genus Babesia.The infection routes in humans are transmitted by ticks or blood transfusion.There are hundreds of Babesia species globally,and only few species of them involved human infection.The infection is asymptomatic in most adult healthy patients,but lethal to those in immunodeficiency condition.So babesiosis poses a high threat to human health.Some researches showed that,of six species that can infect humans,Babesia microti(B.microti)is much more virulent than others.The pathogenicity of B.microti is relatively weak,but it can escape the body’s immune response and could relapse after treatment.Currently,there is no specific therapy for its infection.The clinical diagnosis mainly depends on morphological observation,and immunological and molecular biological detection,and there is no any commercial kit in China yet.However,with the findings of Babesia microti derived antigens for diagnositic purpose,one of them called Babesia microti secreted antigen 1(BmSAl)was believed to have the potential of being as candidate molecule for diagnosis.Full-length BmSA1 containing signal peptide and transmembrane region,is not suitable for soluble expression.In this study,by using biological software,a recombinant truncated BmSA1(tBmSA1)with His tag was expressed in E.coli system,and purified by affinity chromatography.The recombinant protein was proved to be highly specific by indirect ELISA and Western blot.After conjugated to horseradish peroxidase(HRP),tBmSA1was used as detection antigen in double antigen sandwich ELISA system.Another tBmSAl fusion protein,tBmSAl-GST,from Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences was used as the coated protein.After optimization,the experiment condition was finally determined as described below.The concentration of tBmSA1-GST was 6μg/mL,the serum dilution was 1:10,the coating solution was carbonate buffer solution(CBS),the coating condition was 4℃ overnight,the blocking solution was 5%nonfat dry milk,the condition for the blocking is 4 ℃ overnight,the time for the serum incubation is 1h,the dilution of tBmSAl-HRP conjugate isl:1000,the incubation time for HRP labelled antigen is 1h,and the developing time for substrate was 10 min.This system was evaluated by clinical human and animal samples with good result.Of 84 goat serum and 1117 human serums samples,1 from goat and 6 from human was detected positive,and their positivity was confirmed by Western blot.Furthermore,this system showed no any corss reactions to either Schistosoma japonicum,SFTSV,or Toxoplasma gondii,demonstrating good specificity.These positive serums were validated by Western blot,and the results showed bands.So,the ELISA system established in this study is a highly specific and sensitive method for B.microti infection diagnosis.