| Aeromonas hydrophila causes diseases in aquatic organisms such as fish and shrimp.This not only caused serious economic losses to coastal areas,but also greatly restricted the long-term sustainable development of the aquaculture industry.The pathogenicity of A.hydrophila is complex and multi-factorial.It mainly produces and secretes related pathogenic factors,causing the host to get sick.Among them,the ahh1 gene,as a highly toxic pathogenic factor,plays an important role in the host infection process of A.hydrophila.The elucidation of its pathogenic mechanism is of great significance for disease prevention and vaccine development.This article obtained the recombinant protein through the E.coli prokaryotic expression system p ET32a~+,purified by the Ni-NTA resin and obtained the refolded protein through urea gradient dialysis.The study found that the refolding protein can form a hemolysis loop on the blood plate,and the hemolysis rate is up to 170%.The hemolysis rate can be detected when the protein is diluted by 64 times,and the hemolysis rate was18%,indicating that the obtained recombinant protein has high biological activity.The rabbit was immunized with hemolysin and prepared a highly specific rabbit-derived hemolysin polyclonal antibody.After immunizing Cyprinus carpio koi with hemolysin,the serum was collected and the total superoxide dismutases(SOD),lysozyme(LZM)and catalase(CAT)in the serum were tested to explore the immune effect of hemolysin.The results showed that the serum enzyme activities of each immunization group changed significantly in the initial stage of immunization,and reached the maximum value after14 days of immunization.It was found that compared with the control group,SOD activity was 1.9 times higher,LZM activity was 3.8 times higher,and CAT activity was1.7 times higher.Afterwards,the enzyme activity fluctuated but was still higher than the control group,indicating that hemolysin has good antigenicity and can stimulate the host to produce an immune response.In order to further explore the molecular mechanism of hemolysin infecting the host,transcriptome sequencing was performed on the spleen of Cyprinus carpio koi 14 days after hemolysin infection.Differential expression analysis showed that there were 1982significantly differentially expressed genes(DEGs),of which 1083 genes were upregulated and 899 genes were down-regulated.The expression levels of inflammation-related IL-8,TLR5,CTSK,CXCL9 and other genes were significantly up-regulated.Further analysis showed that DEGs were mainly enriched in IL-17 signaling pathway and Toll-like receptor signaling pathway,suggesting that hemolysin stimulation can trigger inflammatory response and activate the immune system.The analysis found that molecules such as MHC1,MHC2 and STAT2 were abnormally down-regulated,suggesting that hemolysin plays a role in evading host immunity.Seven immune-related genes were randomly selected for q RT-PCR experiment to verify the accuracy of RNA-seq data,and the results showed that the expression level of the detected genes was consistent with the sequencing data,indicating that the sequencing data was accurate and reliable.In order to study the function of hemolysin ahh1 gene,the knockout plasmid was constructed using CRISPR/Cas9 technology.Construction of a single plasmid knockout system using BPK764 as a backbone vector,coupled with sg RNA elements and homologous repair arms.The expression plasmid p Target F-ahh1 containing the target site sg RNA was coupled to theλ-Red recombinase system through the homology repair arm using the CRISPR/Cas9 system to construct a double plasmid knockout system.The results of this study constructed a double plasmid knockout system,but the method of transferring the knockout plasmid into the recipient cell and the improvement of knockout efficiency still need further study.The research in this article explained the function of hemolysin in Cyprinus carpio koi’s infection and immunity,clarified the key genes and pathways of Cyprinus carpio koi’s immune response to hemolysin,and constructed a single-plasmid and double-plasmid knockout system for the ahh1 gene.This not only lays a good theoretical foundation for in-depth analysis of the mechanism of hemolysin,but also has significance for the effective prevention and control of diseases caused by A.hydrophila. |
