重组HSP70对罗非鱼外周血淋巴细胞免疫功能的影响

摘要

利用酵母重组分泌表达的罗非鱼HSP70与海豚链球菌抗原在体外非共价结合形成HSP70 -抗原复合物,通过体外增殖试验及免疫相关基因表达检测研究HSP70对罗非鱼外周血淋巴细胞免疫功能的影响.结果表明,tHsp70在体外能够与海豚链球菌菌体、胞外分泌物(ECP)结合形成HSP70-抗原复合物.tHsp70(10 μg/mL)或tHsp70( 10μg/mL) -菌体复合物均可极显著刺激罗非鱼淋巴细胞增殖(P＜0.01),而ECP对淋巴细胞增殖有一定的抑制作用(P =0.056),但tHsp70( 10 μg/mL)与ECP体外非共价结合可减轻ECP对细胞增殖的抑制作用.浓度为100μg/mL的tHsp70或tHsp70-抗原复合物作用于罗非鱼淋巴细胞,ICER、PGK、MMP9、MHCⅡ4个基因的表达量均极显著高于海豚链球菌抗原(菌体或ECP)单独刺激和无刺激对照组(P＜0.01).研究表明罗非鱼HSP70具有免疫增强和免疫佐剂的功能,为开发罗非鱼链球菌病HSP70-肽疫苗提供了试验依据.%In this report,we use the recombinant tilapia Hsp70(tHsp70)from Pichia yeast to prepared non-co valent HSP70-antigen complexes with thallus and extracellular bacteria products( ECP) of Streptococcus iniae, and investigated their effects on cellular immune response of tilapia by detection of Proliferation and related immune gene expression of peripheral blood lymphocyte in vitro. Our results demonstrated that tHsp70 could form complexes with thallus and ECP of tilaplia in vitro. In addition,tHsp70( 10 p,g/mL)and all tHsp70(10 μg/mL)-thallus com plexes enhanced the proliferation of tilapia peripheral blood lymphocytes ( P <0. 01). Similarly, thallus alone also enhanced the proliferation of tilapia peripheral blood lymphocytes ( P < 0. 05 ). By contrast, ECP alone exhibited some inhibitory effect on the proliferation of tilapia peripheral blood lymphocytes( P =0.056). However, the com plex of tHsp70 at 10,but not at 100 and 1 μg/mL,with ECP reduced the inhibitory effect of ECP on the prolifera tion of tilapia peripheral blood lymphocytes. Moreover, tHsp70( 10 μg/mL) alone or tHsp70(10 μg/mL) -antigen complexes could significantly influence the expression of immune related genes(PGK,MMP9,ICER,MHC Df )in ti lapia peripheral blood lymphocyte compare to antigen alone and medium control groups. The study indicated that tH sp70 have the immunoadjuvant and the immunopotentiator function, and provided experimental data for development the tHsp70 based vaccine against tilapia streptococcal diseases.