| Fish farming plays an important role in China’s aquaculture industry,but the increasingly frequent disease problem seriously restricts the sustainable development of fish farming industry.At present,we should resolvethis urgent problem to ensure the quality and safety of aquatic products.In-depth study of the immune mechanism of fish is the fundamental way to achieve this goal.Cynoglossus semilaevis is an important marine aquaculture fish in China.At present,the output value of aquaculture is 10 billion yuan.However,the bacterial and viral diseases cause serious threat to the culture of semi-smooth tongue.The anti-bacterial and antiviral immune defense mechanism is not clear,what’s more,to lack of effective disease prevention and control measures.In view of this,the project was used to study the immune function of CsC8 gene and CsSHP-1 gene which aim to elucidate the molecular mechanism of Cynoglossus semilaevis and provide an important theoretical basis for the disease prevention and treatment of Cynoglossus semilaevis.The specific research contents are as follows:In this study,the CsC8 gene was cloned and verified.The results showed that CsC8 was composed of C8α,C8β and C8γ,which were encoded by CsC8α,CsC8β and CsC8γ,respectively.The CsC8α gene was contained 1797 bp open reading frame(ORF),encoding 598 amino acids.The CsC8β gene was contained 1749 bp open reading frame,encoding 582 amino acids,The CsC8γ gene was contained 666 bp open reading frame,encoding 221 amino acids.The expression of CsC8α,CsC8β and CsC8γin 13 healthy tissues showed that CsC8α,CsC8β and CsC8γ were expressed in differenttissues,the highest in the liver,followed by Spleen and ovary.After infection with Vibrio anguillarum,CsC8α,CsC8β and CsC8γ were expressed in different degrees in the liver,spleen,small intestine,head kidney,gill and blood of six kinds of Cynoglossum semilaevis.The expression of CsC8α in blood(P <0.01).The expression level of CsC8β in head kidney was the most obvious at 12 h,the expression level of CsC8β was 17.8 times(P <0.01)at 12 h,and CsC8γ was also expressed in blood(P<0.01).The inhibitory effect of recombinant protein CsC8α in vitro was the most significant,and the inhibitory effect of recombinant protein CsC8α was the most obvious The inhibitory effect on Edwardsiella and Vibrio parahaemolyticus was enhanced with the increase of protein concentration,although the change trend was weaker than that of eel arc The bacteriostatic effect of bacteria,however In addition to a certain inhibitory effect,when the concentration of recombinant protein exceeded0.3mg/m L,it only reached 0.5 or more,for the Gram-positive bacteria on behalf of Staphylococcus aureus,recombinant protein on its antibacterial effect is not obvious,The bacteriostatic rate was below 0.5,and the inhibitory rate was 0.43 at the highest concentration of 0.5 mg/mL,and no significant bacteriostatic effect was obtained.These results suggest that C8 plays an important role in the immune response,especially for the immune response caused by bacterial pathogens and to the inhibition of Gram-negative bacteria.In this study,the full-length cDNA of CsSHP-1 gene was obtained by RACE and PCR.The full-length cDNA of CsSHP-1 was 2045 bp,and the coding region contained1743 bp,and the coding was 580 amino acids.SMART domain prediction analysis showed that the CsSHP-1 contained two SH2 domains and one PTPc domain.The results showed that the SHP-1 was similar to Oreochromis niloticus.The highest phylogenetic tree shows that fish SHP-1 is first clustered together and then together with higher vertebrates.The expression of CsSHP-1 in 13 healthy tissues showed that CsSHP-1 was expressed in all tissues,but the tissue specificity was obvious,the expression level in spleen was the highest,and the expression in the heart was thelowest(P <0.05).After the infection of Vibrio anguillarum,the expression of different levels of liver,spleen,small intestine,head kidney,gill and blood were different,but the peak time was different.Four kinds of pathogen mimics were used to treat the peripheral After the expression of PolyI: C,the expression of PolyI: C showed a continuous downward trend,showing the characteristic immune response after virus infection.In order to further explore the function of CsSHP-1 in inhibiting tumor activity,we successfully expressed recombinant protein of CsSHP-1 with a molecular weight of 67 kDa in Pichia pastoris eukaryotic expression system.The recombinant protein CsSHP-1 was tested by CCK-8 in vitro cytotoxic activity and intensity of human hepatocellular carcinoma cell line HepG2,human lung cancer cell A549 and human breast cancer cell line MDA-MB-231.The results showed that the recombinant protein CsSHP-1 had different cytotoxic activity on the three kinds of cancer cells,and the order of the cytotoxic activity was human hepatoma cell line HepG2> human breast cancer cell MDA-MB-231> human lung cancer cell A549.In summary,the immune-related genes C8 and SHP-1 play a role in the immune response,and the study of its immune mechanism will lay a theoretical foundation for a more comprehensive understanding of the immune function. |
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