Galectin-1 與 Thioredoxin peroxidase II 基因之選殖及表現;Cloning and expression of Galectin-1 and Thioredoxin peroxidase II genes

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题名:	Galectin-1 與 Thioredoxin peroxidase II 基因之選殖及表現;Cloning and expression of Galectin-1 and Thioredoxin peroxidase II genes
作者:	陳怡均;Yi-Chun Chen
贡献者:	生命科學研究所
关键词:	單株抗體;Galectin-1;Thioredoxin peroxidase II
日期:	2001-01-18
上传时间:	2009-09-22 10:15:51 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	本實驗以RT-PCR的方法合成Gal-1和TPx II的cDNA，並將其構築到 pGEM-T Easy載體上，得到pGEM-Gal I 和 pGEM-TPx II轉殖體；再利用限制酶 (SphI，PstI)切割出Gal-1及TPx II的cDNA片段；並將其接入具有6個組銨酸 (histidine)的表現載體pQE31上，構築成pQE31-Gal I 和 pQE31-TPx II，分別送入大腸桿菌SG13009及XL1-Blue中表現。當以IPTG 誘發出Gal-1和TPx II的表現後，進行蛋白質電泳分析，可在14 KD及22 KD的位置看見明顯的蛋白質表現，再經西方點墨法的分析，進一步證實其為Gal-1及TPx II蛋白。另一方面利用EcoRI自pGEM-Gal I和 pGEM-TPx II切割出Gal-1及TPx II的cDNA片段，並將其接入哺乳類的表現載體pcDNA3.1(+)中，構築成pcDNA3-Gal I及pcDNA3-TPx II；再利用Lipofectamine將這兩個轉殖體分別送入人類的口腔上皮癌細胞 (KB) 及老鼠的纖維母細胞 (3T3)中表現。經G418的篩選，在KB細胞中可得到6株穩定 (stable)的轉殖株，而在3T3細胞中可得到4株穩定的轉殖株，再經西方點墨法的分析，可看見Gal-1及 TPx II蛋白，在穩定轉殖株中的表現量明顯較未轉殖株多。最後，利用Ni-NTA親和性管柱，自含pQE31-Gal I的大腸桿菌SG13009中純化出Gal-1蛋白質，再利用純化出的Gal-1蛋白，當作抗原來免疫Balb/c雌鼠，以便進行單株抗體之製備。經ELISA及西方點墨法的篩選，總共獲得兩株具有分泌Gal-1專一性抗體的融合瘤細胞株 (G1F12B5及G1F12G11)。 cDNAs of Gal-1 and TPx II genes were synthesized using RT-PCR method and constructed in pGEM-T Easy vector for gene cloning. After DNA sequence identification, the Gal-1 and TPx II genes were excised from cloning vector using SphI and PstI enzymes and re-constructed into pQE31 expression vector. Two successful constructs of pQE1-Gal I and pQE31-TPx II were transformed and expressed in E. coli. SG13009 and XL1-Blue, respectively. The results from SDS-PAGE and western blotting showed that 1 mM IPTG induced Gal-1 and TPx II genes expression in E. coli. SG13009 and XL1-Blue in a time dependent manner. Gal-1 and TPx II genes were excised from cloning vector using EcoRI enzyme and re-constructed into another expression vector called pcDNA3.1(+). The constructs pcDNA-Gal I and pcDNA-TPx II were transfected into KB or 3T3 cells using Lipofectamine. By G418 selection, six stable clones in KB cells and four stable clones in 3T3 cells were selected and Gal-1 and TPx II proteins, as indicated by western blot analysis, were overexpressed in these stable clones. The Gal-1 protein was purified by Ni-NTA affinity chromatography, from pQE31-Gal I expressed in E. coli SG13009 and then used as an antigen to immunize Balb/c mice for the monoclonal antibody production. By ELISA and western blotting screening, cloned hybridoma, G1F12-B5 and G1F12-G11, secret the specific antibody against Gal-1 and they were expanded for the monoclonal antibody production.
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