The Antigen Analysis Of Babesia Variabilis And The Optimized Expression Of Recombinant BmSA1 And Its Antigenic Epitope Synthesis

Human babesiosis, an important parasitic zoonosis caused by a blood parasite Babesia microti transmitted mainly through tick bite and blood transfusion.The clinical symptom of Babesia infection varies from an asymptomatic state to malaria-like episodes, and the severe cases could be life-threatening, especially in immunocompromised or elderly patients. At present, Microscopy is still the golden standard in the diagnosis of babesiosis. Due to the low sensitivity of morphological identification and the difficulty to distinguish B. microti from plasmodium, clinical misdiagnosis often occurred and led to delayed treatment. Therefore, it is necessary to develop effective and quick diagnostic methods for babesiosis, for which finding diagnostic antigens including native and recombinant proteins is the key.[Objective]1) To screen effective diagnostic antigens by preparing the crude antigen of B. microti and analyzing antigenic components for establishment of indirect ELISA method for babesiosis; 2) to reduce the cross reactivity of a known antigen Babesia microti secreted antigen 1 (BmSA1) with plasmodium infection and increase the specificity by optimization of its codon and expression of the truncated sequence;3) to identify antigenic epitopes of BmSA1 through predicting and synthesizing the epitope; 4)to produce monoclonal antibodies providing a tool for further development of immunodiagnostics for detection of circulating antigen.[Method]1) The parasites were collected from the blood of BALB/c mice infected with B. microti at the peak of parasitemia. The crude soluble babesia antigen (SBA)was extracted and used to establish an indirect ELISA method. The SDS-PAGE and Western blot were applied to identify immunogenic components; 2) The BmSA1 condon was optimized by software DNA2.0 and the recombinant proteins were expressed in the prokaryotic expression system. The protein obtained was assessed for cross reaction with plasmodium infected human sera ; 3) The reactivity of the expressed proteins of truncated BmSAl sequence at N- terminal and C- terminal was examined and compared with the proteins before truncation for the cross reaction with plasmodium infected human serum; 4) The BmSA1 linear B cell epitope was predicted by using DNASTAR, ABCPred, LEPS and VSMTrip program , and the epitope peptide was synthesized and the diagnostic efficacy was evaluated with the ELISA method established. 5) The BmSA1 protein was used to immunize the mice for production of specific monoclonal antibody.[Result]1) ELISA method based on SBAwas established,which could detect the early Babesia microti infection of mice (at 7d post-infection) with no cross-reaction with the sera of human infected with Plasmodium falciparum, Plasmodium vivax and Toxoplasma, indicating higher sensitivity and specificity.The SDS-PAGE analysis showed 5 main protein bands with molecular mass at 72 kDa, 66 kDa, 60 kDa, 53 kDa and 43 kDa and 7 minor bands at 14.4～116 kDa. The Western blot indicated that the 15 antigen components of the SB A was recognized by the mice serum infected with B.microti, among them 72 kDa, 53 kDa, 43 kDa, 39 kDa and 30kDa proteins showed strong reaction . 2) Soluble BmSA1 protein was expressed efficiently after the codon optimization of the BmSAl gene and showed cross reaction rates of 13.3% and 26.7%with P. falciparum and P. vivax infected human sera, respectively.with. 3) Both truncated recombinant BmSA1 at N- terminal(N-BmSA1) and C- terminal (C-BmSAl)could react with the B.microti infected BALB/c serum. The cross reaction rate of N-BmSAl with babesia serum was the same as that of full length BmSAl, but interestingly, the C-BmSAl had no cross reaction with malaria serum. 4) Five epitopes of BmSA1 at 26-59AA, 239-256AA, 252-271AA, 272-287AA and 281-300AA were identified and showed potential value in immunodiagnosis .The OD value of ELISA for positive mice sera and negative mice sera were 0.211/0.098,1.692/0.235, 0.733/0.169, 0.377/0.078, 0.749/0.275. 5) Three monoclonal hybridoma lines against recombinant BmSAl and soluble crude babesia antigen were obtained.[Conclusions] An indirect ELISA based on crude soluble babesia antigen is established, and could be used as an effective screening tools for P. microti infection.Of the soluble babesia antigen components, 72 KDa, 53 KDa, 43 KDa, 39 KDa-, and 30KDa molecules strongly react with babesia infected mice sera and may be good candidate antigens for immunodiagnosis, however, these immunogenic components remains to be further characterized and evaluated. Compared with the protein of full length of BmSAl gene,the C- BmSAl shows higher specificity, and may be used for diagnosis of B. microti infection. The epitope peptide of BmSA1 provides a base for development of antigen peptide-ELISA diagnostics.