Cloning,Identification,and Antimicrobial Immune Response Of Chemokine CC25-like In Cromileptes Altivelis

Humpback grouper(C.altivelis)is an important marine aquaculture fish in China.The expansion of its aquaculture scale has led to an increasingly serious disease problem.CC chemokines are a class of small cytokines that can promote the migration of immune cells,and they play an important role in fish immunity.In this study,the chemokine CC25-like of humpback grouper(CaCC25-like)gene was cloned by molecular technology and analyzed its disease functions and the results are as follows.Sequences analysis results showed that the open reading frame(ORF)of CaCC25-like was 291 bp and encoded 96 amino acids,mainly including a signal peptide consisting of 20 amino acids and a SCY domain.The results of homology comparison showed that CaCC25-like had higher homology with CC25 of other teleost fish,ranging from 42.45%to 97.92%.Among them,the highest homology with Epinephelus bruneus was 97.92%,and CaCC25-like has a low homology with mammalian CC25,both of which do not exceed 25%.The analysis of phylogenetic tree showed that CaCC25-like belongs to the CCL19/21/25 subgroup.In order to study the function of CaCC25-like,this study analyzed its transcriptional expression level by q RT-PCR.The results showed that in healthy humpback grouper,CaCC25-like was expressed in nine tissues tested.The expression was higher in the intestine,liver,blood,heart.The expression was lower in the spleen,head and kidney,brain and gills.And the expression was lowest in the stomach.After Vibrio harveyi infected humpback grouper,CaCC25-like expression was significantly up-regulated in the spleen and kidney.In order to explore the function activity of CaCC25-like,the prokaryotic recombinant expression plasmid p ET32 a / CaCC25-like was constructed,and the recombinat protein of CaCC25-like(r CaCC25-like)was expressed and purified.In vitro experiments,it showed that when head kidney lymphocytes(HKLs)were incubated with r CaCC25-like,r CaCC25-like could significantly induce lymphocyte migration and cell proliferation with a dose-dependent trend.And r CaCC25-like can significantly inhibit the growth of Streptococcus agalactiae,Vibrio harveyi and Escherichia coli,which indicated that it has the antibacterial ability.In order to explore the antibacterial effect of CaCC25-like,we successfully constructed CaCC25-like overexpression plasmid and synthesized si RNA.In vivo experiments,it showed that when CaCC25-like was overexpressed,the number of bacterial infections significantly decreased in liver,spleen and kidney tissues.When CaCC25-like was unknown,the number of bacterial infections increased significantly in liver,spleen and kidney tissues.In summary,CaCC25-like is a functional chemokine,which has the functions of recruiting lymphocytes,promoting cell proliferation and enhancing host immune defense,and it can play an important role in resisting bacterial infections of humpback grouper.