探討豬細胞激素IL-6與CpG ODN佐劑對豬胸膜肺炎放線桿菌DNA疫苗之效用

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探討豬細胞激素 IL-6 與 CpG ODN 佐劑對豬胸膜肺炎

放線桿菌 DNA 疫苗之效用

指導教授：楊文仁博士國立高雄大學生物科技研究所學生：葉倩如國立高雄大學生物科技研究所摘要 豬胸膜肺炎放線桿菌 (Actinobacillus pleuropneumoniae) 為引起豬隻出血性、纖維 性、壞死性胸膜肺炎之病原，造成全球豬場經濟的重大損失。細胞激素 (cytokine) 具有調節免疫反應之重要角色，利用不同細胞激素刺激可使免疫反應趨向於Th1或Th2之免疫路徑。IL-6 (interleukin-6) 可由單核球、巨噬細胞、纖維母細胞、T及B淋巴細胞等細胞分泌，若以IL-6做為佐劑，可使免疫反應趨向於Th2免疫途徑，促進B細胞產生抗體。CpG ODNs (oligodeoxynucleotides) 為特定DNA序列中含有未甲基化之CpG motif，也是疫苗常用之佐劑，主要是刺激樹突狀細胞分化及誘導 B 細胞、單核球、自然殺手細胞等分泌細胞激素，具有促進免疫之活性。為提升DNA疫苗之效用，本研究以豬IL-6及設計適合豬隻之CpG motif (ATCGAT)，含有不同套數之CpG ODNs序列分別做為ApxIA DNA 疫苗之佐劑，進行動物免疫試驗，檢測此等佐劑對免疫效果之影響。將豬IL-6基因選殖到真核表現載體中建構成，以 DNA 定序確認基因之正確性，並轉染至豬腎細胞 (LLC-PK1) 中，以西方點墨法分析蛋白質，結果pcDNA-IL-6可於真核細胞中表現蛋白質。以MTT檢測法分析IL-6之生物活性，本實驗選殖IL-6 以100 pg/ml刺激NS-1細胞之刺激指數 (stimulation index, SI) 為2.23，市售豬IL-6 SI為2.57，結果顯示，本實驗選殖之IL-6與市售豬IL-6蛋白質皆可刺激NS-1細胞之增生，證實選殖之IL-6具有生物活性。以肌肉注射對小鼠進行免疫試驗，檢測DNA疫苗添加佐劑後之抗體效價，以PBS與單獨施打ApxIA DNA疫苗為對照組，添加IL-6 10g 及50g之免疫劑量，以添加50 g之抗體效價較10g高。含不同套數之CpG ODNs (CpG1~CpG4) 之疫苗佐劑，以CpG 1與CpG 4抗體效價最高。分析IgG抗體亞型之結果，各組以產生IgG2a為主，CpG1、CpG4組之IgG1 及IgG2a皆有增加，且證實可活化Th1細胞與Th2細胞，促進體液性及細胞性免疫反應。。實驗結果證實以添加本實驗設計之CpG 4 (含CpG motif套數最多)之佐劑，可增加ApXIA DNA疫苗之效用

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The Preparation and Effect of Porcine Cytokine IL-6 and

CpG Oligodeoxynucleotides Adjuvant for Actinobacillus

pleuropneumoniae DNA Vaccine

Advisor: Dr. Wen-Jen Yang

Institute of Biotechnology National University of Kaohsiung

Student: Chien-Ju Yeh Institute of Biotechnology National University of Kaohsiung

ABSTRACT

Actinobacillus pleuropneumoniae is the etiological agent that causes haemorrhagic,

fibrinous and necrotic pleuropneumonia in pigs and makes critical economic losses in global pig farms. Cytokines play crucial roles in the regulation of immune responses, and different cytokines can stimulate the immune responses toward to Th1 or Th2 pathway. Interleukin-6 (IL-6) could be produced by variety of cells including monocytes, macrophages, fibroblasts, T cells and B cells. Using IL-6 as adjuvant can promote immune responses toward to Th2 pathway and enhance B cell to produce antibodies. CpG ODNs is a specific DNA sequence contains an unmethylated CpG motif. It plays a critical role in immune activity promotion and stimulating the differentiation of dendritic cells and inducing B cells, monocytes, natural killer cells to secrete cytokines. To improve the efficacy of DNA vaccine, swine IL-6 and various CpG ODNs containing different copy numbers of CpG motif (ATCGAT) which is suitable for swine were designed as the adjuvants for ApxIA DNA vaccine. The adjuvant effects of above designs were evaluated through a series of animal immunization assay. The swine IL-6 gene was amplified by RT-PCR from swine fibroblast cell line (ST) and cloned into eukaryotic expression vector to construct pcDNA-IL-6. The gene sequence was confirmed by DNA sequencing analysis. The pcDNA-IL-6 was transfected into swine kidney cell line (LLC-PK1) and detected the protein expression by Western blot analysis. The results showed that the cloned swine IL-6 can be expressed in eukaryotic cells. The IL-6 bioactivity was measured by MTT assay; the stimulation index (SI) of NS-1 cell line was 2.23 and 2.57 for 100 pg/ml cloned IL-6 and commercial IL-6, respectively. It indicated that both of IL-6 could stimulate NS-1 proliferation and revealed that the cloned IL-6 has bioactivity. To

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explore the antibody responses of DNA vaccine combine with adjuvant, mice were immunized by intramuscular injection. Comparison the adjuvant effect of 10 μg with 50 μg IL-6, the results showed that 50 μg IL-6 induced higher antibody titer. The results of different CpG ODNs adjuvant effect showed that CpG1 and CpG4 elicit the highest antibody titer. In IgG subtypes analysis indicated that IgG2a is the major subtype of the test groups. The IL-6 could induce higher IgG2a than IgG1, however, both of IgG2a and IgG1 were enhanced when administrated with CpG1 and CpG4. It indicated that Th1 and Th2 cells could be activated by CpG1 and CpG4 and promoting cellular and humoral immunity.