點帶石斑魚對小孢子靈芝免疫調節蛋白的免疫反應

Abstract

石斑魚為亞洲地區重要的養殖魚種，但是高密度集約飼養下，卻容易遭受到病毒侵害。為了預防病害的發生，提升魚隻免疫力不失為一有效的策略。本實驗嘗試利用酵母菌生產及純化的重組小孢子靈芝(Ganoderma microsporum)免疫調節蛋白(GMI)使用於點帶石斑魚，來評估其免疫反應。 本研究以GMI，對於點帶石斑魚苗進行腹腔免疫注射後，分別取出頭腎與脾臟兩種免疫器官，再利用即時定量聚合酶鏈鎖反應(Real-time PCR)，分析免疫相關基因的表現量。免疫基因反應的評估則涵蓋先天性免疫與後天性免疫的免疫標的基因。實驗結果發現，GMI能刺激較多在脾臟的免疫基因，證明GMI進入體內後對於不同的免疫器官，其刺激的程度將有所差異。在有效劑量方面，GMI低劑量組別(注射每克魚體2 ng的劑量)，即會刺激IRF3、IRF10、Viperin、ISG15、Mx I、Mx II、CL4、IL-1β和TNF-α等基因表現。而這些免疫基因的調控則與免疫系統中的干擾素傳遞路徑有關。 魚類的干擾素系統，可分為type I inteferon (I-IFN)與type II inteferon (IFN-γ)兩型，影響先天性免疫與後天性免疫反應。GMI皆會刺激這兩型的免疫相關基因表現。Mx為一種受到干擾素調控的基因(IFN-stimulated genes, ISGs)，其在免疫系統中具有抗病毒的能力，點帶石斑的Mx基因可細分為3種不同的亞型(Isoform)；實驗結果發現Mx基因，在魚體內受到GMI刺激時，體內3個Mx基因的表現量會因不同器官和時間而有所差異，此結果暗示這3種Mx基因在免疫系統上各扮演不同角色。 GMI不但具有刺激魚類先天性免疫反應的能力，也能刺激後天性免疫，GMI具有能刺激免疫球蛋白(Igs)的表現能力。本研究探討免疫球蛋白重鍵與輕鍵的基因表現量，發現這些基因受到GMI刺激後，表現量會上升。 在細胞毒性測試中，探討Poly:IC與GMI對於石斑魚頭腎細胞(Grouper Kindey, GK)的影響。將GMI分別以不同劑量配合不同的時間點加入GK細胞中。實驗結果發現，在7.5 μg /ml濃度下12小時，最能刺激GK細胞的免疫基因表現。而為探討GMI是否具有抗病毒的能力，利用石斑魚頭腎細胞，經過8 μg /ml的GMI處理12小時後，再感染石斑魚虹彩病毒，比較同樣濃度Poly:IC處理下，探討石斑魚虹彩病毒基因的表現量變化。發現經過GMI與Poly:IC處理的組別，虹彩病毒的基因表現量都下降，代表兩種處理下都具有能使病毒量降低的能力，且GMI的抗病毒能力優於Poly:IC。

Grouper is an important aquacultural fish species. This particular species has a high commercial value and is widely cultured in Southeast Asia, including in Taiwan. Viral infection can seriously impact the grouper fish industry. An effective strategy to improve grouper fish health is to increase immunity against the pathogen. GMI, an immunomodulatory protein cloned from Ganoderma microsporum, is reported to have the immunomodulatory function. In the present study, we injected and tested the influence of GMI on fish immune system in vivo by measuring the immune gene expression of GMI stimulated grouper head kidney and spleen. The GMI was injected into the Grouper using intraperitoneal injection. Post injection, we used real-time PCR to measure the immune gene expression in its head kidney and spleen. The immune related gene includes the innate immune and adaptive immune system. We observed that GMI had more influence in the spleen. Low dosage of GMI (2 ng/g of fish weight) would stimulate IRF3、IRF10、Viperin、ISG15、Mx I、Mx II、CL4、IL-1β & TNF-α expression which relates to interferon system. The fish interferon system is divided into two group: Type-I Interferon (I-IFNs) and type-II Interferon (IFN-γ). The Fish interferon system involves innate and adaptive immune systems. GMI will stimulate the fish interferon system. Mx, IFN-stimulated genes, can be divided into three isoforms. The gene expression of the profile is different from each other. Evaluated Mx isoforms have a different role in the Grouper immune system. Immunoglobulin M (IgM) is the first antibody that is produced in the immune system and provides a crucial first line of defense for the immune system.. Grouper IgM can be divided to two part, IgM heavy chain (IgM-H) and light chain (IgM-L). We observed that IgM-H and IgM-L genes were up-regulated. It would be prudent to evaluate how GMI can strengthen grouper adaptive immunity. The immune gene in grouper kidney (GK) cell with 7.5 μg /ml GMI can be stimulated to have the most expression. We examined the anti-virus ability of GMI by using GK cells treated with GMI 8 μg /ml at 12 hr then infected with grouper iridovirus (GIV) at 18 hr. The results were compared with Poly:IC. The results showed that GMI and Poly:IC can against GIV. The anti-virus capabilities of GMI was better than Poly:IC. The results show that GMI can not only stimulate the innate immune system but also the adaptive immune system as well.