Preparation And Characterization Of Single Chain Antibodies (scFv) Against Pseudomonas Plecoglossicida

Pseudomonas plecoglossicida is the causative agent of Visceral granulomas disease(VGD)in large yellow croaker(Larimichthys crocea),which can infect a variety of aquaculture economic fish including large yellow croaker,small yellow croaker(Larimichthys polyactis),striped bass(Oplegnathus fasciatus),yellow grouper(Nibea albiflora)and grouper(Epinephelinae).The losses caused by VGD account for about 10%of the total value of large yellow croaker industry,seriously effecting the healthy and sustainable development of the large yellow croaker industry.Therefore,it is necessary to develop an immunological method that is easy to operate,simple to interpret and suitable for rapid diagnosis in the field.In this study,we used the P.plecoglossicida recombinant haemolysin co-regulatory protein(Hcp)as the antigen to screen the specific single-chain antibody(Hcp-sc Fv)from the rabbit natural phage sc Fv library.We constructed the recombinant plasmid p ET-30a-hcp-sc Fv for protein expression to obtain the specific sc Fv against the P.plecoglossicida Hcp.The results laid the foundation for the development of VGD in large yellow croaker.The specific results of the study are as follows.1.Screening of single chain antibodies against P.plecoglossicida Hcp:P.plecoglossicida Hcp(p ET-30a-Hcp)was used as the antigen to screen the target antibodies from a rabbit natural phage single-chain antibody(sc Fv)library by solid-phase antigen screening method.As a result:16 single-chain antibodies(hcp-sc Fv)against P.plecoglossicida Hcp were screened;According to affinity and gene sequence analysis results,seven strains of hcp-sc Fv with high specificity were finally obtained named A5,F11,G5,G8,D2,D10 and H12.2.Preparation of single chain antibodies against P.plecoglossicida Hcp:Specific primers were designed based on the gene sequences of the seven hcp-sc Fv,and the single-chain antibody target gene was amplified by PCR and inserted into the p ET-30a(+)vector to construct a prokaryotic expression plasmid(p ET-30a-hcp-sc Fv).PCR identification and double digestion analysis showed that the seven recombinant plasmids were constructed correctly.The recombinant plasmids were transferred into E.coli BL21(DE3)p Lys S for induced expression.The results showed that the six recombinant strains,A5,F11,G5,G8,D2 and D10,could be stably expressed at a final concentration of 0.4 mmol/m L of IPTG and induced at 30℃for 5 h.The solubility analysis of the recombinant proteins showed that the recombinant proteins of F11,G8,D2 and D10 existed in the form of inclusion bodies,while the recombinant proteins of A5 and G5 existed in both soluble proteins and inclusion bodies.The purified recombinant proteins were immunoreactive against His-Tag monoclonal antibody and the molecular weight of the proteins was around 35 k Da,as expected.3.Development of ELISA for P.plecoglossicida:Two recombinant proteins,A5 and F11,with stable expression,were selected for the establishment of an indirect ELISA for P.plecoglossicida.The results showed that:F11 had a poor affinity for P.plecoglossicida and A5 had a high affinity for P.plecoglossicida;The inclusion condition was 4℃overnight,the dilution of enzyme secondary antibody was 1:2000,the incubation condition of enzyme secondary antibody was 37℃for 1 h,the colour development condition was 37℃for 10min,the threshold value for negative determination was 0.456.The sensitivity and specificity experiments were conducted using the established indirect ELISA method,and the results showed that the sensitivity of the method was 5×10~4cfu/m L,the specificity experiments The results showed that A5 did not cross-react with Nocardia,Edwardsiella retardans and Vibrio parahaemolyticus,but cross-reacted with Vibrio harzianus.In summary,six single-chain antibodies against P.plecoglossicida Hcp were prepared,and an indirect ELISA method was established to detect P.plecoglossicida for two of the single-chain antibodies,A5 and F11.The results showed that the single-chain antibodies can be used to detect the pathogenic bacteria of VGD in large yellow croaker and provided a theoretical basis and foundation for the development of a rapid clinical diagnosis technique for VGD in large yellow croaker.