Preparation Of Polyclonal Antibody Against IgM Of Takifugu Rubripes And Screening And Identification Of Immunogenic Antigens Of Scuticociliates

Takifugu rubripes is an important marine economic animal in northern China.In recent years,the problem of scuticociliates disease has become more and more serious.In this study,we carried out the preparation of IgM polyclonal antibody of T.rubripes,the screening and identification of immunogenic antigens of scuticociliates and the development of anti-ciliate vaccine of T.rubripes.The main results are as follows:1.The CH1-CH4 gene of IgM heavy chain constant region of T.rubripes was cloned and a 1326 bp specific band was obtained by PCR amplification.The prokaryotic expression system of BL21-pET28a-IgM was constructed,and about 62 k Da recombinant protein was obtained.The results of soluble analysis showed that BL21-pET28a-IgM was mainly expressed as inclusion body.BALB/c mice were immunized with the recombinant protein,the specific polyclonal antibody against T.rubripes was successfully prepared.The results of Western blot showed that the antibody could specifically recognize the IgM protein in the serum of natural T.rubripes.2.The polyclonal antibody against Pseudocohnilembus persalinus was successfully prepared using P.persalinus as antigen.The results of DOT-ELISA showed that the titer of the antibody was up to 1:40000;SDS-PAGE and Western blot analysis of specific antibodies showed that the protein at 35 k Da showed the strongest immune response.The protein at35 k Da was isolated,purified and identified by mass spectrometry,the result of mass spectrometry was glyceraldehyde-3-phosphate dehydrogenase(GAPDH).3.The pathogen scuticociliates of T.rubripes in Dalian were sampled,isolated and identified.The pathogen was identified as Uronema marinum by morphological observation and 18 S rDNA sequencing.The isolated pathogen scuticociliates were enriched and cultured in vitro to prepare whole insect vaccine,and CpG ODN was used as immune enhancer to prepare scuticociliate and CpG combined vaccine against T.rubripes.The results showed that the injection of U.marinum whole worm vaccine group compared with the control group,the number of infected scuticociliates of T.rubripes was reduced by 69% within 7 days,the cumulative number of deaths within 14 days was reduced by 50%.The injection of U.marinum whole worm vaccine could effectively prevent scuticociliate infection,while the combined vaccine had no obvious immune effect against scuticociliate infection.The results of ELISA showed that the serum IgM expression levels of T.rubripes in the experimental group reached the peak on the 14th day,and the serum IgM expression levels in the whole scuticociliate vaccine group were the highest,while the serum IgM expression levels in the control group had no overall change,indicating that vaccine injection can effectively stimulate the immune response of T.rubripes.This study provided a tool for the immunological experiment based on IgM of T.rubripes,provided a reference for the immunogenicity antigen analysis of scuticociliates,and provided a thought for the immunological prevention and treatment of scuticociliates.