Molecular Identification Of Haptoglobin From Japanese Sea Bass (Lateolabrax Japonicus) And Its Application In The Diagnosis Of Infection With Vibrio Harvey

Haptoglobin（Hp）is mainly synthesized by liver and plays an important role in acute phase response（APR）induced by infection,injury and inflammation.In this study,the full-length c DNA sequence of Hp gene（Gen Bank No.MK820673）was obtained from the transcriptome of liver from Japanese sea bass（Lateolabrax japonicus）.Lj Hp consists of 2285 nucleotides which possesses one large open reading frame（ORF）,which is predicted to encode a 309 amino acids precursor with a calculated molecular weight of about 33.82 k Da.The N-terminal 22residues are the signal peptide.Sequence analysis showed that Lj Hp had the typical characteristics of fish Hp,and shared the highest amino acid homology with that of European sea bass（Dicentrarchus labrax）（76.8%）,followed by swamp eel（Monopterus albus）Hp with 75.7%homology;phylogenetic tree analysis showed that fish Hps formed a large cluster,and Lj Hp and Hps from swamp eel,large yellow croaker（Larimichthys crocea）,zebra mbuna（Maylandia zebra）,greater amberjack（Seriola dumerili）,gilthead seabream（Sparus aurata）and the European sea bass（Dicentrarchus labrax）grouped into a small cluster,among which Lj Hp is most closely related to European sea bass and swamp eel Hps.Lj Hp m RNA is mainly expressed in the liver of healthy Japanese sea bass.After infection with Vibrio harveyi,the expression of Lj Hp m RNA in the liver,spleen and head kidney of Japanese sea bass upregulated significantly（P<0.05）.The mature peptide of Lj Hp gene was successfully expressed in prokaryote and its antiserum was prepared.Western blot analysis showed that Lj Hp native protein was modified by N-glycosylation in liver and serum of Japanese sea bass,and its level increased significantly in serum after V.harveyi infection（P<0.05）.The above results showed that the expression of Lj Hp m RNA and its protein are closely related to V.harveyi infection.It is suggested that Lj Hp might be a positive acute phase protein（PAPP）involved in the resistance of Japanese sea bass to bacterial infection.The recombinant Lj Hp protein（r Lj Hp）was obtained by nickel column purification and urea gradient refolding.Based on this,the enzyme-linked immunosorbent assay（ELISA）for the development of Lj Hp content in Japanese sea bass serum was determined.The direct coating method had a higher linear correlation than the double antibody sandwich method.The best dilution ratio of Lj Hp antiserum was 1:80000.The best coating time was 12 h.The best reaction time of the first antibody and the second antibody were 2 h and1 h,respectively.The best incubation time for color development was 15 min.The sensitivity of the method is 32 ng/m L,and the standard curve was obtained,and the linear regression equation is y=0.001x+0.0179（R²=0.9909）,where x and y represent r Lj Hp concentration and OD₄₅₀value respectively.With this method above,Lj Hp content in serum of Japanese sea bass was evaluated.The level of Lj Hp in sera of Japanese sea bass was significantly higher than that of the control group at 6 h post infection（hpi）,reached the peak at 12 hpi,and gradually decreased thereafter,but still significantly higher than that of the control group at96 hpi.The results of our study not only provide the basis for further research of the function of Hp in fish and its molecular mechanism in APR caused by pathogenic bacteria,but also provide molecular markers and technical support for the evaluation of fish infection.