Study On The Antigenicity And Immunoregulatory Property On Dendritic Cells Of Ts-Sp-7 Protein Of Trichinella Spiralis

Trichinella spiralis is a typical zoonotic parasitic nematode worm that causes Trichinosis,a foodborne parasitic disease that is widespread worldwide.At present,the main diagnostic methods are artificial digestion and ELISA.Excretion and secretion products(ES)are the most widely used as diagnostic antigens,however,their application is limited due to their phase-specificity and complex components.ES can trigger host Th2 immune response,resulting in host immunosuppression,thus forming chronic infection and leading to long-term parasitism.The mechanism of this reaction is still unclear at this stage.The ES components of Trichinella Spiralis are complex and contain many proteases,including serine protease,glutathione transferase,cysteine protease,aspartic protease and metalloproteinase.Therefore,the isolation,identification,antigenicity and immunoregulatory studies of Trichinella Spiralis ESassociated antigen molecules are very important for the establishment of immunological detection methods and the development of vaccines.The c DNA library of Trichinella spiralis 3-day adult worms was constructed in our laboratory in the early research,and the serine protease gene Ts-Sp-7 with high abundance and strong antigenicity was obtained through immunological screening(Gen Bank registration number EU263332.1).In this study,the antigenicity and immunoregulatary property of this protein were studied from the following three aspects:Firstly,specific primers were designed according to the Ts-Sp-7 gene sequence published on the Genbank.The total RNA of the Trichinella spiralis adult stage of infection for 3 days was collected.Reverse transcription and PCR amplification were proceeded to obtain the target sequence.The prokaryotic expression vector p ET28a-TsSp-7 for prokaryotic expression was constructed.SDS-PAGE result showed that a protein band was appeared at about 47 k Da,which was consistent with the theory size of 47.5 k Da.The renatured and purified Ts-Sp-7 protein was harvested.Western-Blot result showed that the target protein produced a specific response to the positive serum of pigs with different infection days(15 dpi,30 dpi,45 dpi,60 dpi,90 dpi,120 dpi)with a dose of 10000 / head.The results showed that the target protein had good reactogenicity.Secondly,the denatured purified Ts-Sp-7 protein was used as the immunogen to prepare rabbit polyclonal antibodies against Ts-Sp-7 protein.The titer of the polyclonal antibody was 1:350000 by indirect ELISA.As can be seen from the indirect immunofluorescence results,compared with the rabbit negative serum control group,the experimental group with Ts-Sp-7 protein polyclonal antibody as the primary antibody showed a strong red fluorescence signal on the skin of muscle larvae.Meanwhile,denatured purified Ts-Sp-7 protein was used as immunogen,and the renatured purified recombinant Ts-Sp-7 protein and ES were used as the screening agents to screen the hybrid tumor cell lines that could stably secrete anti-Ts-Sp-7 antibody.Through three subclones,5 hybridoma cell lines were harvested,which could stably secrete anti-Ts-Sp-7 protein antibody.Competitive ELISA results showed that the pig positive serum could inhibit the binding of two strains of monoclonal antibodies to recombinant Ts-Sp-7 protein,so both strains were competitive antibodies,named as H4H7-2A4 and H8D12-5B9.Western-Blot results showed that both H4H7-2A4 and H8D12-5B9 can specifically identify the crude antigen.The subtype identifications showed that heavy chain type of H4H7-2A4 was Ig G2 a,light chain type is ?;heavy chain type of was Ig G2 b,light chain type is ?.In this experiment,the sequence of TsSp-7 gene was used as the template,and three pairs of primers were designed to construct the recombinant expression vectors.Then the Ts-Sp-7 protein was expressed in three segments.Western-Blot results showed that both strains of monoclonal antibodies specifically identified Ts-Sp-7 protein.Furthermore,8 small peptides were synthesized by Pep Scan technology and then tested by indirect ELISA.By the epitope identification,H4H7-2A4 against antigen epitope was in W5 peptide,and the amino acid sequence was 222GVDRSATCQGDSGGP236.H8D12-5B9 against antigen epitope was in W7 peptide,and amino acid sequence was 252PPTCGDARHSVKFAKVP268.The successful preparations of polyclonal antibodies and specific competitive monoclonal antibodies provided the basis for the establishment of methods for the diagnosis of Trichinella spiralis infection based on Ts-Sp-7 protein.Finally,dendritic cells(BMDCs)were isolated and extracted from bone marrow derived cells of mice,which were induced to differentiate in vitro to form immature BMDCs.Ts-Sp-7 protein separate function group,LPS positive control group and the negative control group were set up and analysised the effect of Ts-Sp-7 protein on BMDCs by flow cytometry.The results showed that the Ts-Sp-7 protein induced the expression of BMDCs surface molecule CD86,and there was no obvious effect on the expression of MHC-?.These results indicated that Ts-Sp-7 protein promoted the maturity of BMDCs.Na?ve CD4+T cells were isolated and extracted from the spleen of OVA antigen specific transgenic mice(OT-II),and a co-culture system was established with pre-stimulated BMDCs by Ts-Sp-7 protein.Under the condition of OVA antigen stimulation,the activation,proliferation and differentiation of Na?ve CD4+T cells were tested.The results showed that Ts-Sp-7 protein obviously promoted the proliferation of Na?ve CD4+T cells.It promoted the expression of Na?ve CD4+T cells surface effector CD44 and CD62,and promoted the activation of effector T cells into effector T cells that participate in lymphocyte recycling.The BMDCs incubated with Ts-Sp-7 protein induced Na?ve CD4+T cells to differentiate into Treg cells,suggesting that Ts-Sp-7 protein was a recombinant protein with immunoregulation and involved in host immune regulation.The results indicated that Ts-Sp-7 protein had good antigenicity and immunoregulatory property,which can be used as diagnostic antigen,and play an important role in the regulation of host immune response as an important regulatory molecule of Trichinella spiralis.