- 學位論文
茯苓免疫調節蛋白之基因選殖及其對類異位性皮膚炎老鼠模式之效應
Molecular Cloning of the Immunomodulatory Protein PCP and its Effect in Atopic Dermatitis-Like Mouse Model
摘要
茯苓 (Poria cocos) 免疫調節蛋白 PCP，是分子量為 35.6 kDa 的醣蛋白，已知可藉由 TLR4-MyD88 途徑活化小鼠腹腔巨噬細胞，另外在有 anti CD3/CD28 存在下，PCP可活化 CD4+ 與 CD8+ 細胞的增生，故 PCP 為一具有免疫調節活性潛力的物質。為了進一步了解 PCP 的免疫調節機制與生化活性，故本研究分為兩個部分，第一部分欲選殖 PCP 的基因序列以幫助了解 PCP 的生化特性或其活性位置；第二部分則是利用類異位性皮膚炎小鼠動物模式，瞭解餵食 PCP 是否會減緩過敏小鼠的症狀。在第一部分的結果，根據 PCP 的 N 端胺基酸序列合成 PCP 之 degenerate 引子，再以 3’ 及 5’ Rapid amplification cDNA end (RACE) 的方法，選殖出全長為 807 bp 的 PCP cDNA，此序列的 ORF 為 579 bp，轉譯後可推衍出 194 胺基酸，預測分子量為 12.765 kDa。將此序列轉形至大腸桿菌株表現出 PCP 重組蛋白 (His)6-PCP，再利用 anti-His 與 anti-PCP 抗體進行西方轉漬法分析，兩種抗體皆可在 17 kDa 到 21 kDa 之間辨認到條帶。在第二部分試驗結果，餵食 PCP 組能降低淋巴結細胞分泌 IL-4 與 IL-5 的分泌量，但 IFN-γ 含量因故無法判定；此外餵食 PCP 會降低致敏鼠血清中的 OVA-specific IgG1 含量，同時增加血清中 OVA-specific IgG2a 的含量。在血清中 OVA-specific IgE 含量檢測，與正控制組相比，餵食 PCP 組能降低其含量。故推測餵食 PCP 處理可降低 OVA 致敏所引起的體內 Th2 免疫反應。綜合以上結果，本研究選殖出 PCP 的基因序列，與初步推測在類異位性皮膚炎動物模式中，餵食 PCP 可抑制致敏鼠體內免疫平衡趨向 Th2 免疫反應，改善過敏情況。
並列摘要
Poria cocos immunomodulatory protein, PCP, was a glycoprotein with a molecular weight of 35.6 kDa. In murine models, PCP not only could activate primary macrophages through TLR4-MyD88 pathway, but also could enhance the proliferation of CD4+ and CD8+ cells in the presence of both CD3/CD28 antibodies. These studies showed that PCP was a potential immunomodulatory protein. The objectives of this study were first, to clone the cDNA of PCP, and second, to use an atopic dermatitis-like (AD-like) model to investigate whether administration of PCP could down-regulated the disease symptoms of mouse. In the first part, we designed the PCP degenerate primers based on their N-terminal amino acid sequence. Using the 3’ and 5’ Rapid amplification cDNA end (RACE) method, the cDNA sequence of PCP was cloned. The full length of PCP cDNA was 807 bp, and the ORF contained 579 bp nucleotide acids, encoding 194 amino acids. Predicted molecular weight of ORF encoding amino acid was 12.765 kDa. The ORF sequence obtained from PCP cDNA was further transformed into an E. coli expression system to produce PCP recombinant protein, (His)6-PCP. In the Western blotting analysis with anti-His and anti-PCP monoclonal antibodies, both antibodies recognized (His)6-PCP between the molecular weight 17 kDa to 26 kDa, hence to confirm the accuracy of PCP. In second part, oral administration of PCP suppressed the levels of IL-4 and IL-5 in draining LNs. However, the expression of IFN-γ was failed to be detected in the draining LNs. Oral administration of PCP suppressed the level of serum OVA-specific IgG1 but up-regulated the level of IgG2a. In the case of OVA specific IgE, oral administration of PCP could suppress IgE secretion in comparison with the positive control. These results suggested that the oral administration of PCP could suppress the OVA-induced Th2 response. Taken together, we this study cloned the cDNA of PCP and suggested that PCP could inhibit OVA-induced Th2 response to suppress a symptom of atopic dermatitis.