摘要
制备鳗弧菌(Vibrio anguillarum)全菌灭活疫苗浸泡免疫牙鲆(Paralichthys olivaceus),分别于免疫后0h、4h、8h、12h、24h、48h、72h、96h、7d、14d取脾、头肾、鳃组织,提取m RNA,应用实时荧光定量PCR法检测3种组织中Toll-like受体(TLR)2、TLR5M、髓样分化因子My D88、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、热休克蛋白(HSP)70、T细胞表面分子CD4、自然杀伤细胞增强因子(NKEF)十一种免疫相关基因的表达变化。结果显示,免疫后除TLR5M、NKEF以外其它九种基因表达均显著上调,表达高峰出现在24—72h,基因表达量最高值是对照组的2—12倍;TLR5M的表达量与对照组无显著差异;NKEF基因的表达量出现显著下调趋势,下调峰值出现在24h,为对照组的0.49倍。在脾脏和肾脏中,NF-κB和CD4基因的表达峰值高于鳃;在脾脏和鳃中,IL-6、HSP70和NKEF基因的表达峰值均高于头肾;IFNγ、CXC、C3和My D88在三个组织中的表达峰值差异不大。在三个组织中每个基因表达峰值出现的时间基本一致。结果表明,浸泡免疫后,IL-6和HSP70基因在三个组织中的表达变化迅速、且丰度高,可以作为疫苗浸泡免疫后的效果评价指标;除肾和脾主要的免疫器官外,鳃也是浸泡免疫后重要的检测组织。研究结果为浸泡免疫疫苗效果的评价积累了数据。
We prepared inactivated whole-cell vaccine of Vibrio anguillarum, and then vaccinated Paralichthys olivaceus in whole-cell vaccine immunization. Samples of spleen, head kidney and gill of P. olivaceus were collected at 0h, 4h, 8h, 12 h, 24 h, 48 h, 72 h, 96 h, 7d, and 14 d after immunization. Total RNA was extracted from spleen, head kidney and gill, and then the gene expression levels of TLR2(Toll-like receptor 2), TLR5 M, Myeloid differentiation factor 88(My D88), NF-κB(nuclear transcription factors), IL-6(interleukin-6), IFNγ(interferon γ), CXC(chemotactic factor), C3(complement factor), Hsp70(heat shock protein 70), cluster determinant 4(CD4) and nature killer enhancing factor(NKEF) were tested in q RT-PCR(quantitative reverse transcription-polymerase chain reaction). The results show that the expression quantities of immuno-related genes increased significantly after immunization except for TLR5 M and NKEF. The maximum expression of immune-related genes occurred in 24~72h after immunization, and was 2~12 times higher than those of control group. However, TLR5 M showed no significant difference; and the expression of NKEF showed down-regulation and reached peak at 24 h after immunization. The minimum expression was 0.49 times of control group. In spleen and head kidney, the maximum expression of NF-κB and CD4 was higher than that in gill. In spleen and gill, the maximum expression of IL-6, HSP70, and NKEF was higher than that in head kidney. The maximum expression of IFNγ, CXC, C3, and My D88 occurred in similar time and showed no significant difference in the three tissues. The fast change and high expression of IL-6 and HSP70 in all the three tissues may be applied for evaluation on fish soaking vaccine immunity. In addition to the main immune organs including spleen and head kidney, gill can be an important tissue for the detection. This method may be applied for future evaluation on the efficacy of vaccination and its molecular mechanism.
引文
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