貓腸上皮細胞的原代培養(yǎng)及cDNA文庫的構建
發(fā)布時間：2019-03-21 06:51
【摘要】：目的建立貓腸上皮細胞(Intestinal epithelial cells,IECs)培養(yǎng)體系,構建其酵母雙雜交cDNA文庫,為篩選弓形蟲毒性因子在終末宿主的互作蛋白奠定基礎。方法分別采用組織塊法以及低濃度膠原蛋白酶Ⅺ和中性蛋白酶Ⅰ聯(lián)合消化法進行貓IECs的原代培養(yǎng),通過形態(tài)學觀察及細胞免疫組織化學方法鑒定后提取mRNA,運用SMART技術合成cDNA第一鏈,以LD-PCR擴增獲得雙鏈cDNA(ds cDNA),通過同源重組方法在酵母菌株Y187中構建貓IECs的cDNA文庫,計算文庫的容量,以酵母菌液PCR檢測插入片段的大小分布。結果兩種培養(yǎng)方法效果表明酶聯(lián)合消化法更為有效。本研究建立了連續(xù)培養(yǎng)貓IECs體系,培養(yǎng)出高純度的貓IECs,用其構建的cDNA文庫容量為1.1×10~6,滴度為2.8×10~9cfu/ml,插入片段大小在0.5~2.0 kb之間。結論培養(yǎng)的原代貓IECs構建的cDNA文庫符合酵母雙雜交篩選互作因子標準,這為篩選弓形蟲終末宿主的毒性因子互作蛋白奠定了基礎。
[Abstract]:Aim to establish a culture system of cat intestinal epithelial cells (Intestinal epithelial cells,IECs) and construct a yeast two hybrid cDNA library for screening the interaction proteins of toxoplasma toxoplasmosis toxic factors in the terminal host of Toxoplasma gondii (Toxoplasma gondii). Methods the primary culture of cat IECs was carried out by tissue block method and low concentration collagenase XI and neutral protease 鈪，
本文編號：2444695
[Abstract]:Aim to establish a culture system of cat intestinal epithelial cells (Intestinal epithelial cells,IECs) and construct a yeast two hybrid cDNA library for screening the interaction proteins of toxoplasma toxoplasmosis toxic factors in the terminal host of Toxoplasma gondii (Toxoplasma gondii). Methods the primary culture of cat IECs was carried out by tissue block method and low concentration collagenase XI and neutral protease 鈪，
本文編號：2444695
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