Study On Immunogenicity Of Myophilin? LDH Protein In Echinococcus Multilocularis Protein And Detection Of Circulating Nucleic Acids

Objective: 1.The present study aims to clone and express myophilin and LDH genes of Echinococcus multilocularis,and identify the immunogenicity of myophilin and LDH recombinant protein,which Lays a theoretical foundation of the immune diagnosis research of Echinococcus multilocularis infection based on myophilin and LDH proteins.2.The present study aims to detecte specific miRNA and DNA of Echinococcus multilocularis in serum of alveolar echinococcosis patients and use them as biomarkers of worm body infection,which provides theoretical foundation of research on measuring method of Echinococcus multilocularis infection based on circulating nucleic acids.Methods: 1.Myophilin and LDH genes were cloned by PCR technology and linked into pET15 b vector.Recombinant expression vectors were constructed and transformed into E.coli Rosetta(DE3)competent cells.Recombinant proteins were induced and expressed.Expression forms of recombinant proteins were detected by SDS-PAGE.Recombinant proteins were purified by affinity chromatography of Ni-IDA resin.Immunogenicity of recombinant proteins were identified by Western blotting.Serum samples from patients with alveolar echinococcosis(57 cases),cystic echinococcosis(33 cases),and healthy persons(50 cases)were examined by ELISA,which evaluated preliminarily immune diagnosis effect of recombinant proteins.The B and T cell antigen epitopes of ecombinant proteins were predicted using the DNAstar software.2.The expression stasus of miRNA in serum of alveolar echinococcosis patients was analyzed by high throughput sequencing in order to determine specific miRNA of Echinococcus multilocularis in serum.The DNA in serum of alveolar echinococcosis patients was detected by PCR in order to determine specific DNA sequences of Echinococcus multilocularis in serum.Results: 1.Myophilin and LDH genes were cloned successfully and the corresponding recombinant proteins were expressed and purified.Results of Western blotting showed myophilin and LDH recombinant proteins were recognised by serum samples from patients with alveolar echinococcosis and cystic echinococcosis,but not by serum samples from healthy persons.Results of ELISA showed that diagnostic sensitivities of myophilin recombinant protein reacted with serum samples from patients with alveolar echinococcosis and cystic echinococcosis were 80.70% and 78.79% respectively.Diagnostic sensitivities of LDH recombinant protein reacted with serum samples from patients with alveolar echinococcosis and cystic echinococcosis were 84.21% and 84.85% respectively.Prediction results of DNAstar showed myphilin protein might have 8 B cell antigen epitopes.They were located at 15-29?41-48?72-77?110-115?130-136?145-161?169-177 and 181-188 amino acids,respectively.myphilin protein might have 6 T cell antigen epitopes.They were located at 46-53?59-75?82-96?100-108?117-123 and 155-163 amino acids,respectively.LDH protein might have 4 B cell antigen epitopes.They were located at 52-57?81-87?97-102 and 221-227 amino acids,respectively.LDH protein might have 3 B cell antigen epitopes.They were located at 39-45?76-86 and 225-235 amino acids,respectively.2.Three possible miRNA of Echinococcus multilocularis were screened by high throughput sequencing.They were emu-miR-124 a,emu-miR-87 and emu-miR-9,respectively.But when compared with human mi RBase,their homologies were 100%.They didn't have specific.Specific DNA sequences of Echinococcus multilocularis in serum haven't been detected by PCR after screening 48 primers.Conclusion: 1.Myophilin and LDH recombinant proteins of Echinococcus multilocularis have high immunogenicity and good immune diagnostic value for echinococcosis.2.Specific miRNA and DNA of Echinococcus multilocularis were not detected in serum samples from patients with alveolar echinococcosis.