Chemotactic Activity And Immune Adjuvant Effect Of Grass Carp Beta Defensin 1

Grass carp（Ctenopharyngodon idella）is the most prominent large-scale freshwater economic fish cultured in China,and its output has been steadily rising in recent five years.It has always occupied the first position in production.All kinds of bacterial diseases that occur in grass carp culture seriously restrict the healthy development of grass carp culture.Antibiotics are often used to deal with grass carp bacterial diseases in intensive farming.Still,the abuse of antibiotics is easy to causes bacterial resistance crisis,causing harm to the environment and public health.β-defensin is a small cysteine-rich cationic peptide that plays a significant role in the host’s innate immune defense.β-defensin exists widely in all kinds of organisms,and its anti-bacterial activity has been widely studied.Further studies showed thatβ-defensin not only plays a significant role in the battle against pathogenic microorganisms and has a variety of immune regulatory activities,playing a crucial role in congenital and adaptive immunity.In this study,the anti-bacterial function of grass carpβ-defensin 1（CiBD1）was verified,and its chemotaxis activity and adjuvant effect on A.hydrophila inactivated vaccine were explored.It lays an excellent theoretical foundation for applying CiBD1 in production practice.Relevant experimental results are as follows:1.CiBD1 is a class of cationic amphiphilic proteins with highly conserved threeβ-folded structures.By Clustal Omega multi-sequence alignment and I-TASSER secondary structure prediction,we revealed that CiBD1 is a small cysteine-rich cationic peptide with amphiphilic properties.Its structure is highly structured and conserved in teleost fish.2.CiBD1 is constitutively distributed in all tissues of grass carp and highly expressed in some mucosal tissues.In order to explore the physiological role of CiBD1,we studied the transcription level of CiBD1 gene in different tissues of grass carp under normal physiological conditions,and the results showed that the skin and eye were the two tissues with the highest level of CiBD1 transcription before infection.The transcription level of CiBD1 in all tissues of grass carp infected with A.hydrophila at 72 h was significantly different from that before infection,which showed that the transcription level of CiBD1 in tissues related to mucosal immunity,such as skin,eyes,swim bladder and hindgut,was significantly higher.3.CiBD1 recombinant protein has antimicrobial activity on common pathogenic bacteria of fish and Ichthyophthirius multifiliis.The prokaryotic expression vector p ET-32a-CiBD1 was constructed and induced by IPTG.The recombinant CiBD1 protein was purified by Ni²⁺affinity chromatography and digested by enterocyte kinase.The anti-bacterial activity of CiBD1 recombinant protein was verified by CFU plate method.It has significant inhibitory activity against G⁺and G^-.CiBD1 recombinant protein showed concentration-dependent bactericidal activity against trophozoites,and 70%of trophozoites could be killed by 40μmol/L of CiBD1 protein for 20 min.4.CiBD1 recombinant protein has leukocyte chemotactic activity,which can enhance the protective effect of A.hydrophila inactivated vaccine on grass carp.Chemotactic experiments showed that CiBD1 showed the best chemotactic activity against primary white blood cells of grass carp at 50 ng/m L.The adjuvant immune effect of CiBD1 recombinant protein on A.hydrophila inactivated individual experiments investigated vaccine.The group’s mortality rate with CiBD1 adjuvant was below the contrast group and vaccine group,and the levels of Ig M and MHCⅡtranscription and the contents of complement C3 and lysozyme in serum were significantly higher than those of the control group.In conclusion,CiBD1 is a class of highly conserved small-molecule cationic antimicrobial peptides.CiBD1 is distributed in various tissues of grass carp in a constituent form and is highly expressed in some mucosal tissues.When pathogens or environmental stimuli stimulate the organism,CiBD1 transcription level expression in some tissues in close contact with the peripheral environment is more significant.CiBD1 recombinant protein had particular killing activity against bacteria and Ichthyophthirius multifiliis in vitro and showed solid chemotactic activity against lymphocyte and myeloid cells of grass carp.CiBD1 recombinant protein can improve the protection rate and disease resistance of A hydrophila inactivated vaccine and have an excellent immune adjuvant effect.This study indicated that CiBD1 recombinant protein plays a vital role in bacterial anti-infection immunity and has a particular application prospect in aquaculture.