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標題: 製備犬IGFBP5重組蛋白質以偵測乳腺癌患犬血清之抗IGFBP5自體抗體
Preparation of the recombinant canine insulin-like growth factor-binding protein 5 (IGFBP5) to detect anti-IGFBP5 autoantibodies in sera of dogs with mammary carcinoma
作者: 潘韻安
Yun-An Pan
關鍵字: 犬乳腺腫瘤
canine mammary gland tumors
recombinant protein
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摘要: 犬乳腺瘤是母犬最常發生的腫瘤。透過定量蛋白質體學的分析,我們實驗室先前已找出類胰島素生長因子結合蛋白5 (insulin-like growth factor-binding protein 圓石5 ,簡稱IGFBP5) 於乳腺癌組織中過量表現,並以西方墨點法作確認。本研究進一步以免疫組織化學染色確認,IGFBP5表現於乳腺癌組織的上皮細胞。IGFBP5為一種參與調控細胞的生存與凋亡的分泌型蛋白,已被視為多種人類癌症「腫瘤相關抗原」。腫瘤相關抗原可能誘發「自體抗體」的產生;而自體抗體,可作為腫瘤早期診斷的生物標記。本研究目的為製備犬IGFBP5重組蛋白,以偵測乳腺腫瘤患犬血清中之抗IGFBP5自體抗體。研究中構築了兩個表現載體:pET30a(+)-IGFBP5 (嵌入全長IGFBP5基因片段) 與pET24a(+)-His-IGFBP5∆1-23 (嵌入去除信號肽的IGFBP5基因片段),以表現同時在N端及C端帶有組氨酸標籤 (His-tag) 的犬IGFBP5重組蛋白。將上述表現載體送入大腸桿菌BL21(DE3)或C41(DE3),以異丙基-β-D-硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,簡稱IPTG) 誘導犬IGFBP5重組蛋白表現後,再以鎳離子親和層析法來純化重組蛋白。實驗結果顯示,IGFBP5∆1-23重組蛋白在BL21(DE3)的表現量及純度較高,故純化IGFBP5∆1-23重組蛋白作爲抗原,以年齡相仿的乳腺癌早期病犬 (2隻)、與晚期病犬 (2隻)、與健康犬 (1隻) 之血清進行西方墨點法分析。結果顯示,和正常犬血清相比,乳腺癌患犬的血清針對IGFBP5∆1-23重組蛋白具較顯著的免疫反應。本研究為首次確定犬血清中含有抗IGFBP5自體抗體,且該抗體有潛力成為診斷犬乳腺瘤的工具。
Canine mammary tumors (CMTs) are the most common type of neoplasia in female dogs. Our lab has previously identified overexpression of insulin-like growth factor-binding protein 5 (IGFBP5) in canine mammary cancers and has verified its elevated protein level in cancerous tissues by Western blotting. In this study, the elevated IGFBP5 expression in epithelial component of mammary carcinoma was further examined by immunohistochemistry (IHC). IGFBP5, a secreted protein involved in regulation of cell survival and apoptosis, has been identified as a tumor-associated antigen (TAA) in various types of human cancer. TAAs are likely to elicit autoantibodies (AAbs) which hold potential as tumor biomarkers for early detection. The present study aims to express the recombinant canine IGFBP5 protein to access the presence of anti-IGFBP5 AAbs in sera of CMT dogs. Two expression vectors, pET30a(+)-IGFBP5 and pET24a(+)-His-IGFBP5∆1-23, were constructed for expression of both N- and C-terminally His-tagged recombinant canine IGFBP5 and its truncated form lacking a signal peptide (amino acid 1-23), respectively. Expression of recombinant His-IGFBP5-His or His-IGFBP5∆1-23-His was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli BL21(DE3) or C41(DE3), followed by purification of recombinant proteins using Ni-NTA affinity purification methods. As it was purified in a better yield and purity, the recombinant His-IGFBP5∆1-23-His was used as the antigen to assess seral IGFBP5 AAbs by Western blotting. Five serum samples collected from age-matched female dogs, including 2 dogs with early-stage CMT, 2 dogs with late-stage CMT, and 1 healthy donor, were applied for the Western blot analysis. As a result, sera collected from CMT dogs were highly reactive to the recombinant IGFBP5∆1-23 protein compared with the healthy control. This study reveals for the first time that IGFBP5 AAbs are accessible in canine sera which are likely useful for detection of CMT.
文章公開時間: 2021-08-16
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