台灣金線連免疫調節蛋白基因選殖及表現之研究

Abstract

台灣金線連 (Anoectochilus formosanus Hayata) 是具有台灣地域意義的保健植物，本研究室先前已純化發現金線連免疫調節蛋白 IPAF，分子量為 14 kDa，等電點為 5.4。IPAF 可透過細胞表面受器 TLR4 活化小鼠巨噬細胞，不需要 T 淋巴細胞即可直接活化 B 淋巴細胞，也推測 IPAF 屬胸線非依賴性第一型抗原 (type 1 thymus-independent antigen)，本研究的目的為選殖 IPAF 的基因。首先利用 N 端胺基酸定序分析取得 IPAF 部分胺基酸序列 ASSLGTGGTLRNN，並依結果設計專一性 degenerate 引子，及依序利用 3’-RACE 及 5’-RACE 技術進一步選殖取得 IPAF cDNA，分析IPAF 之核甘酸與胺基酸序列，並與 NCBI 資料庫比對是否有相似序列。結果顯示IPAF cDNA全長為654-bp，其 ORF 為 474-bp，ORF 可轉譯 158個胺基酸，經SignalP prediction 程式分析得到前面的第 1~25 個胺基酸為 signal peptide，經 compute pI/Mw tool 程式計算第 26~158個胺基酸序列，推測其分子量為 14312.02 Da，pI 值為 9.62，且其分子量與天然純化之 IPAF 分子量相近。至NCBI 資料庫比對 IPAF 胺基酸序列結果顯示，IPAF 與蘭科植物 Epipactis helleborine lectin 具有高度相似。進一步將 IPAF 的 cDNA 構築於 pET30-IPAF 表現載體，以大腸桿菌為宿主進行融合蛋白，經 1 mM IPTG 誘導 6 個小時可以大量得到重組 IPAF。本研究選殖出的 IPAF 胺基酸序列，未來可更進一步探討 IPAF 之蛋白質結構、活性部位及保健功效。

Some previous studies have showed that IPAF (immunodulatory protein of Anoectochilus formosamus) could activate mouse macrophages through TLR4 pathway and directly stimulated the proliferation of B lymphocytes without the help from T lymphocytes. These results suggested that IPAF might be a type 1 thymus-independent antigen. The objective of this study was to clone the cDNA encoding IPAF. First, we used the N-terminal amino acid sequence assay to acquire the N-terminal amino acid sequence of IPAF, ASSLGTGGTLRNN, and designed the IPAF degenerated specificity primers based on the N-terminal amino acid sequence. We then used 3’-rapid amplification of cDNA ends (RACE) and 5’ RACE PCR to generate the full-length IPAF cDNA. We then searched in NCBI database for similar nucleotide and amino acid sequences. The result showed that the full-length IPAF cDNA was 654-bp, and ORF was 474-bp, respectively. The protein encoded consisted of 158 amino acid residues. It was predicted that IPAF contained a putative residual signal peptide of 25 residues by the SignalP prediction assay. The molecular weight and pI value of amino acid sequence from residue 26 to 158 as predicted by the compute pI/Mw tool assay were 14312.02 Da and 9.62 respectively, which were similar to the native IPAF. The result of cross searching in NCBI database showed that the amino acid sequence of IPAF was highly similar to that of Epipactis helleborine lectin. A cDNA encoding mature IPAF was cloned into pET30 plasmid vector and expressed in Escherichia coli.. A fusion protein could then be induced by 1mM IPTG for six hours. This study suggested that the amino acid sequence of IPAF provided supportive information for future investigation regarding the structure, active domain and functional analysis of IPAF.