草鱼β-防御素1的趋化活性及免疫佐剂效应研究  

CHEMOTACTIC ACTIVITY AND IMMUNE ADJUVANT EFFECT OF GRASS CARPΒETA DEFENSIN 1

作　　者：况睿 周建成 刘小玲[1] 苏建国 袁改玲[1] KUANG Rui;ZHOU Jian-Cheng;LIU Xiao-Ling;SU Jian-Guo;YUAN Gai-Ling(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Wuhan Dabeinong Aquatic Science and Technology Co.Ltd,Wuhan 430090,China)

机构地区：[1]华中农业大学水产学院,武汉430070 [2]武汉大北农水产科技有限公司,武汉430090

出　　处：《水生生物学报》2022年第12期1855-1866,共12页Acta Hydrobiologica Sinica

基　　金：中央高校基本科研业务费专项基金(2662019PY068)资助。

摘　　要：为了探究草鱼防御素的免疫调节作用,研究通过Clustal Omega多序列比对和I-TASSER二级结构预测,发现草鱼β-防御素1(Ctenopharyngodon idellaβ-defensin 1,CiBD1)是一类具有两亲性的富含半胱氨酸的小分子阳离子肽,其结构在硬骨鱼类中高度保守;通过构建pET-32a-CiBD1原核表达载体,IPTG诱导表达后经过Ni^(2+)亲和层析法纯化和肠激酶酶切获得CiBD1重组蛋白,通过CFU平板法验证了CiBD1的抑菌活性,当蛋白浓度达到200μg/mL时,对革兰氏阴性菌(G+)及革兰氏阳性菌(G–)都具有显著的抑制活性;趋化实验表明CiBD1在50 ng/mL时对草鱼原代白细胞趋化活性最佳;通过个体实验探究了CiBD1重组蛋白对嗜水气单胞菌灭活疫苗的免疫佐剂效应,发现添加CiBD1佐剂组死亡率显著低于单独疫苗组,且该组IgM和MHCⅡ转录水平及血清中补体3(C3)和溶菌酶含量都显著高于对照组。研究表明CiBD1重组蛋白在细菌抗感染免疫中发挥着重要作用,在水产养殖中具有一定的应用前景。β-defensin(BD)is a small cationic peptide rich in cysteine,which plays an important role in host defense and innate immunity.BDs exist widely in all kinds of organisms in nature,and were initially concerned and studied extensively due to its spectral antibacterial activity.With in-depth research,it has been found in recent years that BD not only has direct antiviral microbial activity,but also plays an important regulatory role in natural immunity.At present,there are few studies on the immunomodulatory function of fish defensin.The study on grass carp BD still remained in the analysis of antibacterial activity.In this study,the antibacterial function of grass carpβ-defensin 1(CiBD1)was verified,and its chemochemogenic activity and adjuvant effect on Aeromonas hydrophila inactivated vaccine were explored,which can provide new insights for the prevention and treatment of grass carp bacterial disease.Relevant experimental results are as follows:1.CiBD1 is a class of cationic amphiphilic proteins with highly conserved threeβ-folded structures.Through Clustal Omega multi-sequence alignment and I-Tasser secondary structure prediction,it is found that CiBD1 is a kind of amphiphile cationic peptide rich in cysteine.Its structure is highly conserved in bony fishes.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 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 Aeromonas hydrophila at 72h 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.The recombinant CiBD1 protein was succ

关 键 词：Β-防御素 抗菌活性 趋化活性 免疫佐剂效应 草鱼 

分 类 号：S941.4[农业科学—水产养殖]