HER2/Neu蛋白相對應抗體之製備及研究

HER2/Neu，為第二型人類表皮生長因子接受體。在臨床上，約有10-25%的乳癌患者具有HER2/Neu基因的過度表現情形，而且往往與腫瘤的轉移以及容易復發有關，荷爾蒙療法以及傳統的化學療法對於這些患者的治療效果都很差。現今的檢測方式主要以免疫組織化學染色法以及螢光原位雜交法來偵測HER2/Neu表現情形，但免疫組織化學染色法專一性不高，而螢光原位雜交法雖然較為精確，但檢測時間長，因而開發一種準確且快速的檢驗試劑是有其必要性的。本實驗中，我們利用polymerase chain reaction (PCR)來放大六段不同大小片段的HER2/Neu基因，不過其中只有HER2-618片段於轉殖到大腸桿菌後可以被大量地純化出來，雞隻與兔子在注射這些純化後的重組HER2-618蛋白後，分別產生非常好的IgY與IgG抗體反應，這些多株抗體利用western blot、ELISA以及Immunofluorescence staining也都顯示它們對於HER2/Neu蛋白有特異性的結合能力。為了提高將來發展檢驗試劑的特異性，我們也利用phage display antibody的技術，建構了兩個分別具有1.2 x 107與5.6 x 106含有short或long linker的antibody libraries，並且進一步地，利用此兩個基因庫來篩選出對HER2/Neu具有特異性結合的單株抗體。由於目前已經有研究指出，HER2/Neu是可以利用患者血清來檢測其在體內表現情況，因此，我們希望可以利用此多株及單株抗體來發展血清免疫之檢驗試劑，期望未來可以早期發現乳癌患者是否有HER2/Neu過度表現的情形，以協助醫師用以評估後續診斷及治療方式，進而提高患者存活率。

關鍵字

乳癌抗體；噬菌體展示法；單鏈變異片段抗體

並列摘要

HER2/Neu is the type two of Human Epidermal growth factor 2. In clinical, there are approximately 10–25% patients of breast cancer would over-express HER2/Neu gene. It’s also related to the tumor metastasis and recurrence. For the therapy, both the hormone treated and traditional chemotherapy are poor and limited. Today, the ways to detect the expression of HER2/Neu are immunohistochemical (IHC) and fluorescent in situ hybridization (FISH). However, the specificity of IHC is not well. And FISH would take a long time to analysis even its specificity is better. Thus, it is important and needed to generate the accurate and rapid diagnostic reagent. In this study, we constructed six different recombinant DNAs and transformed into E. coli after amplification of 6 different fragments by PCR. There was only the HER2-618 could be expressed and purified various protein. After the chicken and rabbit immunized with HER2-618 recombinant protein respectively, it succeed to generate the polyclonal chicken IgY and rabbit IgG respectively. Both the two polyclonal antibodies could specifically bind to the HER2/Neu protein in western blot, ELISA and immunofluorescence staining analysis. In order to enhance the specificity for the diagnostic reagent, we constructed two libraries of short linker 1.2 x 107 and long linker 5.6 x 106 antibody library. After bio-panning, we selected monoclonal antibodies with specific binding ability from the two antibody library. There are some reports indicted the HER2/Neu protein will release into blood to detect the expression level. For this reason, we hope both the polyclonal and monoclonal antibodies would be used to develop the diagnostic reagent which could be used to detect early whether HER2/Neu have over-expression or not for the breast cancer patients. It would enhance the survival rate of patients by helping physicians to evaluate and select therapy ways.

並列關鍵字

HER2/Neu ； HER2 antibody ； phage display ； scFv

參考文獻

1. Muller, V. and K. Pantel, HER2 as marker for the detection of circulating tumor cells. Breast Cancer Res Treat, 2009. 117(3): p. 535-7.

3. Komoto, M., et al., HER2 overexpression correlates with survival after curative resection of pancreatic cancer. Cancer Sci, 2009. 100(7): p. 1243-7.

4. Linderholm, B., et al., Shorter survival-times following adjuvant endocrine therapy in oestrogen- and progesterone-receptor positive breast cancer overexpressing HER2 and/or with an increased expression of vascular endothelial growth factor. Med Oncol, 2009. 26(4): p. 480-90.

6. Amodio, R., et al., Target therapy in HER2-overexpressing breast cancer patients. OMICS, 2011. 15(6): p. 363-7.

8. Ross, J.S., Breast cancer biomarkers and HER2 testing after 10 years of anti-HER2 therapy. Drug News Perspect, 2009. 22(2): p. 93-106.

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HER2/Neu蛋白相對應抗體之製備及研究

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