福尔马林灭活柱状黄杆菌对草鱼免疫相关基因表达的影响
EFFECT OF FORMALIN INACTIVATED FLAVOBACTERI UM COLUMNARE ON EXPRESSION OF IMMUNE RELATED GENES IN GRASS CARP, CTENOPHARYNGODON IDELLUS
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摘要: 在本研究中,将福尔马林灭活的柱状黄杆菌菌苗(FKG4)经腹腔注射免疫草鱼,以注射灭菌PBS作对照,分别在免疫后1、7、15和28d,提取受免鱼和对照鱼肝脏、脾脏和头肾3种组织中的总RNA并反转录成cDNA,利用Real-time PCR方法对不同组织中C-反应蛋白(CRP)、主要组织相容性复合体I(MHCⅠ)、肿瘤坏死因子(TNFα)、白介素1(IL-1β)、白介素8(IL-8)、Ⅰ型干扰素(IFNⅠ)等6种免疫相关基因的表达进行定量分析。结果发现CRP在受免鱼肝脏中的表达于免疫后1、7d显著高于对照鱼,而在脾脏与头肾中的表达未出现显著差异;MHCⅠ在受免鱼3种组织中的表达于免疫后1、7、15d都显著高于对照鱼;TNFα在3种组织中的表达水平都较低,但都于免疫后1、7d显著高于对照鱼;IL-1β在3种组织中的表达于免疫后1d都显著高于对照鱼,但只有在头肾中的表达在免疫后7d时仍保持显著差异;IL-8在3种组织中的表达都只是于免疫后1d显著高于对照鱼;TNFα、IL-1β及IL-8等3种炎症因子的基因表达在免疫后15d都恢复到对照鱼的水平;而IFNⅠ在3种组织中的表达与对照组之间未出现显著差异。结果表明,FKG4注射免疫可以显著提高草鱼3种组织中与抗菌免疫相关的基因表达,从而增强鱼体抵抗细菌性病原的免疫力。Abstract: Immune responses of fish are normally regulated by various cytokines and their encoding genes,which are generally called immune related genes.Expressions of different immune related genes of fish were previously proved to be effected by vaccination with various pathogens,including bacteria,viruses,parasite,and so on.In the present study,the expressions of six immune related genes in liver,spleen and head kidney of grass carp,i.e.C-reactive protein(CRP),major histocompability complex I(MHC I),tumor necrosis factor α(TNFα),interleukin-1β(IL-1β),interleukin-8(IL-8) and interferon I(IFN I) as well,were quantified using quantitative real-time polymerase chain reaction at 1,7,15 and 28 days post intraperitoneal injection(dpi) with formalin killed Flavobacterium columnare G4(FKG4),respectively.Control fish were injected intraperitoneally with sterile phosphate buffer solution(PBS).The gene expression of CRP in liver of grass carp was detected at a higher level than that in either spleen or head kidney of fish,and was elevated significantly by vaccination with FKG4 at 1 and 7 dpi,with the highest expression being observed at 1 dpi.By contrast,the gene expression of CRP in either spleen or head kidney of fish was detected at a relatively low level,with no significant difference being observed between vaccinated and control fish at all four detected time points.Significant higher gene expression of MHC I was observed at 1,7 and 15 dpi in all three detected tissues of vaccinated fish when compared with control fish,while its gene expression at 28 dpi showed no significant difference between vaccinated and control fish.Gene expression of TNFα in all three detected tissues of fish was detected at a quite low level,with a significant higher gene expression being observed at 1 and 7 dpi in detected tissues of vaccinated fish compared with control fish,and its gene expression in three tissues of vaccinated fish at 15 and 28 dpi are at the same level as that of control fish.Gene expressions of IL-1β in liver and spleen of vaccinated fish were significantly enhanced at 1 dpi and then down-regulated to control level at 7,15 and 28 dpi,while its gene expression in head kidney was significantly elevated at 1 and 7 dpi and then decreased to control level at 15 and 28 dpi.Gene expressions of IL-8 in three tissues of vaccinated fish were significantly up regulated at 1 dpi,and then down regulated to control expression level in all detected tissues at 7,15 and 28 dpi.However,the gene expression of IFN I,on the other hand,did not change significantly in all three detected tissues of vaccinated fish at four time points,although a slight increase of gene expression of IFN I in these tissues was also noticed at 1,7 and 15 dpi.It is suggested that vaccination of FKG4 be rather effective on increasing expressions of immune related genes in grass carp,which are mainly related to the fish immunity against infection of bacterial pathogen.
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