Screening Of The Membrane Protein Of The Snail Antigen Of Eriocheir Sinensis And Its Virulence Function Of ORF0313 Protein

The Chinese mitten crab, Eriocheir sinensis, is an economically important freshwater species for aquaculture in China. With the development of intensive aquaculture, various disease caused by bacteria and other pathogens have emerged,and started to threaten the sustainability of the aquaculture populations of E. sinensis frequently. Tremor disease (TD) is one of the most serious diseases in E. sinensis and Spiroplasma eriocheiris has been proved to be the cause of TD. A lot of research related to S. eriocheiris has been done, such as pathogen detection, genome sequence and so on. However, few studies have been done on the pathogenic proteins of S.eriocheiris. In the present study, the immunogenic proteins identificed from membrane proteins of S. eriocheiris. The gene and protein expressions as well astoxicity of ORF0313 protein were studied in the present research. This study will provide a theoretical basis for the infection mechanism and the prevention of the pathogen. This study includes the following three aspects:1. Screening of antigenic membrane proteins by immunogenic proteins32 proteins and 6 proteins (2 proteins in common) in S. eriocheiris were screened by anti-S. eriocheiris serum and negative serum. 30 proteins in S. eriocheiris were identified relation with the virulence. 30 proteinsincluded the recognized outer membrane and theproteins: pyruvate, kinase, putative enolase, molecular chaperone DnaK, pyruvate dehydrogenase E2, and glyceraldehyde 3-phosphate dehydrogenase,are involved in infection.2. ORF0313 protein was analysed and studied bybioinformatics analysis,cloning, site-directed mutagenesis and expressionThe signal peptide, transmembrane helix region, structure of ORF0313 protein were analyzed by bioinformatics. It is speculated that the ORF0313 protein may be a membrane protein or secreted protein. The ORF0313 gene was cloned by the genome of S. eriocheiris, which contains 1091 bp basic group. Theoretical molecular weight and isoelectric point for ORF0313 are 39.5 kDa and 4.9. After cloning ORF0313,site-directed mutagened twice, in order to make TGA change into TGG. After transcribe recombinant plasmid into E. coli ( BL21), recombinant protein ORF0313 was expressed and the purified.3. The antibody preparation, localization oftarget celland the cytotoxicity of ORF0313 proteinAfter immunizing the rabbits using the recombinant ORF0313, the anti-ORF0313 serum was prepared. ORF0313 proteins were present in the membrane and cytoplasm by Western blot analysis. Further, the ORF0313 protein was localized in the cell membrane by the colloidal gold mark. The experiment of cytotoxicity of ORF0313 protein showed that activity of E. sinensis hemocytes was increased by CCK-8 Kit. The results showed that the ORF0313 protein has an important correlation with the S .eriocheiris infection to hemocytes of E. sinensis.