CAJ | 학술논문

制备鳗弧菌(Vibrio anguillarum)全菌灭活疫苗,采用腹腔注射和浸泡两种方式分别免疫牙鲆(Paralichthys olivaceus),于免疫后0 h、4 h、8 h、12 h、24 h、48 h、72 h、96 h、7 d、14 d分别取牙鲆脾、头肾、鳃3种组织,提取mRNA,应用实时荧光定量PCR法检测3种组织中白介素(IL)-1β、肿瘤坏死因子(TNF)α、主要组织相容性复合体(MHC)MCHⅠ、MHCⅡ、T细胞表面受体CD4、T细胞表面受体CD8、免疫球蛋白(Ig)M 7种免疫相关基因的表达。结果显示,两种方式免疫后各基因表达量均出现显著上调。注射组3种组织中, IL-1β、TNFα基因的表达高峰出现时间在12~24 h, MHCⅠ、MHCⅡ、CD4、CD8的表达高峰出现在48~72 h, IgM的表达高峰出现在免疫后7 d,各基因表达量最高值是对照组的2~70倍；浸泡组3种组织中, IL-1β、TNFα基因的表达高峰出现在免疫后12~48 h, MHCⅠ、MHCⅡ、CD8表达高峰出现在48~96 h,但CD4在头肾和鳃中表达高峰出现在72 h,在脾中表达高峰出现在7 d, IgM在鳃中表达高峰出现在96 h,在脾和头肾中表达量在14 d内逐渐上升,各基因表达最高值是对照组的2~20倍。结果表明,注射组各组织基因的转录水平均高于浸泡组,且脾、头肾中表达量最高值出现时间早于浸泡组,但鳃中各基因峰值出现时间晚于浸泡组。研究结果为疫苗的免疫途径及免疫效果评价提供了基础数据。
제비만호균(Vibrio anguillarum)전균멸활역묘,채용복강주사화침포량충방식분별면역아평(Paralichthys olivaceus),우면역후0 h、4 h、8 h、12 h、24 h、48 h、72 h、96 h、7 d、14 d분별취아평비、두신、새3충조직,제취mRNA,응용실시형광정량PCR법검측3충조직중백개소(IL)-1β、종류배사인자(TNF)α、주요조직상용성복합체(MHC)MCHⅠ、MHCⅡ、T세포표면수체CD4、T세포표면수체CD8、면역구단백(Ig)M 7충면역상관기인적표체。결과현시,량충방식면역후각기인표체량균출현현저상조。주사조3충조직중, IL-1β、TNFα기인적표체고봉출현시간재12~24 h, MHCⅠ、MHCⅡ、CD4、CD8적표체고봉출현재48~72 h, IgM적표체고봉출현재면역후7 d,각기인표체량최고치시대조조적2~70배；침포조3충조직중, IL-1β、TNFα기인적표체고봉출현재면역후12~48 h, MHCⅠ、MHCⅡ、CD8표체고봉출현재48~96 h,단CD4재두신화새중표체고봉출현재72 h,재비중표체고봉출현재7 d, IgM재새중표체고봉출현재96 h,재비화두신중표체량재14 d내축점상승,각기인표체최고치시대조조적2~20배。결과표명,주사조각조직기인적전록수평균고우침포조,차비、두신중표체량최고치출현시간조우침포조,단새중각기인봉치출현시간만우침포조。연구결과위역묘적면역도경급면역효과평개제공료기출수거。
Vaccination is an effective method for controlling bacterial and diseases of fish. At present, researchers have paid more attention to variations of immune related genes expression to evaluate the immune effect of vaccine, besides the changes of antibody level of serum and mucus, enzyme activity, number of lymphocytes, respiratory burst, com-plement activity and so on. The proinflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor α(TNFα) and chemokines are the key factors in inducing inflammatory and immune cell migration, immune regulation and stimulating the proliferation of T cells and B cells. Major histocompatibility complex class I (MHC I), major histo-compatibility complex class II (MHC II), T cell surface receptor CD4, T cell surface receptor CD8 and immunoglobulin M (IgM) play key roles in different stages such as presentation and recognition of antigens and immune response after pathogens invasion. These factors finally to remove pathogens and maintain healthy through a series of immune re-sponse. Numerous studies have showed that the vaccine could cause rapid changes in many immune related genes. For purpose of studying the influence of immune methods of vaccine on expression of immune related genes, Paralichthys olivaceus were injected intraperitoneal and immersed with inactivated Vibrio anguillarum vaccine respectively in this study. The expression of seven immune related genes, IL-1β, TNFα, MHC I, MHC II, CD4, CD8 and IgM in spleen, head kidney and gills was determined by quantitative real-time polymerase chain reaction (qRT-PCR) at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 7 d and 14 d post immunization. The results showed that, the expression of all the tested genes varied significantly. In the injection group, the expression of IL-1βand TNFαreached the peak between 12–24 h, and MHC I, MHC II, CD4 and CD8 occurred between 48–72 h and peak of IgM appeared at 7 d. The gene maximum ex-pression was 2–70 times higher than that of control group. By contrast, in immersion group, the expression of IL-1βand TNFαreached their peak between 12–48 h, and MHC I, MHC II, CD4 and CD8 occurred within 48–96 h. IgM peak appeared at 96 h in the gills, but 14 d later in spleen and head kidney, the maximum amount was 2–20 times higher than that in control. The results indicated that, the transcriptional level of genes in injection group was higher than that of immersion group in spleen and head kidney. In addition, the highest value appeared earlier in injection group than in immersion group. However, the gene expression peak in gills appeared later than immersion group. Data are referring for the evaluation of vaccine.