摘要
为制备鳗弧菌（Vibrio anguillarum）全菌灭活疫苗,采用腹腔注射的方式免疫牙鲆（Paralichthys olivaceus）。于免疫后0、4、8、12、24、48、72、96h和7、14d取脾、头肾、鳃组织,提取mRNA,应用实时荧光定量PCR法检测3个组织中Toll-like受体（TLR）2、TLR5M、髓样分化因子MyD88、核转录因子（NF）-κB、白介素（IL）-6、干扰素（IFN）γ、趋化因子CXC、补体C3、C型溶菌酶（C-Lyz）、热休克蛋白（Hsp）-70、主要组织相容性复合体MHC II、T细胞表面受体CD4和膜结合型免疫球蛋白（mIg）D这13种免疫相关基因的表达。研究显示：注射免疫后,3个组织中,除了mIgD和TLR5M基因,其余11种基因与对照组相比均呈上调表达,上调表达高峰均在12~72h,基因表达量最高值是对照组的2~30倍;mIgD基因的表达显著下调或无显著差异,基因表达量最低值出现在8~12h,最低值是对照组的0.02~0.2倍;TLR5M基因的表达无显著变化。在头肾和脾脏组织中,除了TLR5M、CXC、C-Lyz和mIgD 4个基因其余9个基因的表达量显著高于鳃组织,且表达量最大值的出现时间均早于鳃组织。研究结果表明：腹腔注射免疫鳗弧菌全菌灭活疫苗能引起牙鲆脾脏、头肾和鳃免疫相关基因的快速变化。脾脏、头肾中基因的表达量大多显著高于鳃组织,且表达高峰值的出现早于鳃组织。注射免疫后,脾脏或头肾组织中TLR2、NF-κB和IL-6基因的表达量上调较快、较高,可作为注射免疫鳗弧菌全菌灭活疫苗效果的主要评价指标。本研究为牙鲆疫苗免疫效果的评价提供了理论依据。
Vaccination is an effective method of controlling bacterial diseases of fish.At present,researchers have paid more attention to the variation of immune related gene expression to evaluate the immune effect of vaccine,then to the changes of antibody level of serum and mucus,enzyme activity,number of lymphocytes,respiratory burst,and complement activity among others.For studying the influence of vaccine immune ways on the expression of immune related genes,Paralichthys olivaceus was intraperi toneally injected with inactivated Vibrio anguillarum vaccine in this study.The spleen,head kidney and gill were taken at 0,4,8,12,24,48,72,96 hand 7,14 dafter immunization,respectively,and then total mRNA was extracted and reversely transcribed into cDNA.The expression of 13 immune related genes including the toll-like receptor（TLR）2,TLR5 M,myeloid differentiation factor 88（MyD88）,nuclear transcription factor（NF）-κB,interleukin-6（IL-6）,Interferon（IFN）γ,chemokines,C-type lysozyme,heat shock protein（HSP）70,major histocompatibility complex class II（MHC II）,T cell surface receptor CD4 and immunoglobulin D（mIgD）genes was analyzed with quantitative real-time polymerase chain reaction（qRT-PCR）using 18 sRNA as internal reference.The results showed that,the expression of all the tested gene except for TLR5 M and mIgD was up-regulated significantly.The expression of TLR2,MyD88,NF-κB,MHC II,CD4,IL-6,IFNγ,CXC,C3,C-Lyz and HSP70 genes maximized between 12 h and 72 h,2~30folds higher than that of control.The expression of mIgD gene was down-regulated but not significantly different from that of control,peaked between 8hand 12 hwith the maximum 0.2~0.02 fold higher than that of control.The expression of TLR5 Mgene was similar in all three tissues.Our findings indicated that,the abundance of TLR2,MyD88,NF-κB,MHC II,CD4,IL-6,IFNγ,C3,and HSP70 gene transcripts in spleen and head kidney was higher than that of these genes in gills.In addition,the highest expression reached earlier in spleen than in gill
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2016年第2期42-53,共12页
Periodical of Ocean University of China
基金
国家重点基础研究发展计划(2012CB114406)
国家科技支撑计划(2012BAD17B02)
国家自然科学基金项目(31172429
31302216)
泰山学者工程专项经费项目资助~~
关键词
牙鲆
鳗弧菌
灭活疫苗
注射免疫
免疫相关基因
Paralichthys olivaceus
Vibrio anguillarum
inactivated vaccine
injection
immune related gene
