| 摘要: | 異烯醇酶，也稱為磷酸丙酮酸水合酶，是種在許多生物種都豐富表達的細胞質蛋白之一，它會催化2 - 磷酸甘油酸變成磷酸烯醇丙酮酸，是分解代謝糖類途徑最後步驟的的關鍵糖解酵素酶。
在腫瘤細胞中，異烯醇酶可促使細胞經由糖解作用進行厭氧增殖（又稱Warburg效應），有時表現在細胞表面，進而促使癌細胞的侵襲。在本文中，我們利用大腸桿菌表現和純化hENO1重組蛋白，將其表現與大量地純化出來，用這些重組的hENO1重組蛋白免疫雞隻。經過四次免疫過後，產生高效價的IgY 抗體反應，將多株抗體稀釋到32000倍皆還有反應。這些多株抗體利用 Western blot、ELISA偵測也都顯示它們對於hENO1 蛋白有特異性的結合能力。接著從免疫過後雞隻的脾臟B細胞，製作針對hENO1的單株抗體，利用噬菌體展示技術建立了兩種重組抗體片段(single chain variable fragment, scFv)基因庫(library)。我們也利用噬菌體展示技術構建兩個抗體基因庫short linker和 long linker分別具有7.8×107與5.4×107 的複雜度，並且進一步地，利用此兩個基因庫來篩選出對hENO1有特異性結合的單株抗體。經過三輪篩選選擇，再隨機挑選表達單株抗體，定序出來的結果short和 long linker皆篩出七群單株抗體。接著用西方墨點法篩選出可以辨認細胞hENO1單株抗體後，以流式細胞儀及螢光免疫染色法進行單株抗體的結合能力測式，其中以hEn S7及hEn S8結合能力最好。在細胞生長抑制實驗也可以發現，此兩株單株抗體對肺癌細胞A549和PE089及肝癌細胞HepG2215生長確實有抑制的效果，經過四天作用hEn S7抑制率分別最高可達46.2%、45.6%、60%，hEn S8抑制率分別最高可達54.9%、58.4%、68.3%。最後以NOD/SCID小鼠進行動物模擬實驗，也發現其篩出之單株抗體可以抑制小鼠體內肝癌細胞HepG2215的生長速率，抑制率最高可以達50%左右。總之，我們認為這些IgY多株抗體和scFv單株抗體可能可以應用在分子診斷劑和治療劑的開發，在未來可以對相關的癌症有幫助。
Enolase, also known as phosphopyruvate hydratase, was discovered in 1934 by Lohman and Mayerhof. It is one of the most abundantly expressed cytosolic proteins in many organisms. It is a key glycolytic enzyme that catalyzes the dehydratation of 2-phosphoglycerate to phosphoenolpyruvate, in the last steps of the catabolic glycolytic pathway.
In tumor cells, expressed enolase at surface is not only upregulated and supports anaerobic proliferation but also promotes cancer invasion especially enolase. And it is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. In this study, we generated polyclonal IgY and single-chain variable fragment (scFv) anti-enolase antibodies from immunized chickens. The E. coli-derived recombinant alpha-enolase protein was purified to its high homogenicity as verified by SDS-PAGE. After the 5th immunization, a high titer of specific polyclonal anti-alpha-enolase antibodies and was elicited in immunized chickens and specifically recognized the purified human alpha-enolase antigen as determined by Western blot and ELISA. After the 5th immunization specfically bind to α-enolase when titered at 1 : 32000X dilution.The expressed heavy and light chain variable genes (VH and VL) were isolated from spleen B cells and amplified to construct phage antibody libraries containing scFv molecules. We constructed two scFv antibody libraries with short and long linker were 7.8 x 107 and 5.4 x 107 transformates, respectively. After 3 rounds of panning, scFv-expressed clones were analyzed, sequenced and purified in advance. They were classified into 7 groups from short linker and 7 groups from long linker. Then, we used western blot to screen the binding ability of monoclone and both NSCLC (A549 and PE089) and HCC (HepG2, Hep3B and HepG2215) cell lysates. After flow cytometry and IFA analysis, hEn S7 and hEn S8 scFv have the best combination of capability between NSCLC (A549 and PE089) and HCC (HepG2215) cells. In vitro, to determine whether hEn S7 and hEn S8 scFv could inhibit NSCLC and HCC tumor growth, we performed cell growth inhibition assays on NSCLC (A549 and PE089) and HCC (HepG2215) cells. After four days treatment, hEn S7 can inhibit respectively 46.2%, 45.6%, 60% of cell proliferation, hEn S8 can inhibit respectively 54.9%, 58.4%, 68.3% of cell proliferation. In vivo, hEn S7 and hEn S8 scFv exhibited inhibition of HepG2215 cell xenograft tumor proliferation rate in NOD/SCID mice. The highest inhibition rate can reach about 50%. All together, it is believed that these IgY and scFv antibodies have potential to develop diagnostic agents and cancers therapy in the future. |
