血液因子動(dòng)物生物反應(yīng)器研制與產(chǎn)品開發(fā)課題報(bào)告

趙雅琳 曾凡偉

摘 要：該項(xiàng)目旨在利用乳腺生物反應(yīng)器成功獲得目標(biāo)蛋白的基礎(chǔ)上進(jìn)行深入研究，綜合應(yīng)用分子生物學(xué)、細(xì)胞學(xué)、胚胎學(xué)、繁殖學(xué)、育種學(xué)、生物化學(xué)、藥理學(xué)等多學(xué)科手段研究動(dòng)物生物反應(yīng)器所生產(chǎn)藥物蛋白的有效性和安全性。將圍繞目標(biāo)蛋白的臨床前研究中的中試批量質(zhì)控生產(chǎn)、藥理毒理學(xué)等進(jìn)行研究，從而使國(guó)內(nèi)首個(gè)轉(zhuǎn)基因動(dòng)物生產(chǎn)的新型生物藥品的開發(fā)邁進(jìn)重要一步。該研究課題獲得了高表達(dá)且穩(wěn)定遺傳的牛和羊乳腺生物反應(yīng)器，且抗凝血酶III轉(zhuǎn)基因陽(yáng)性羊種群規(guī)模已達(dá)81只，轉(zhuǎn)基因牛種群規(guī)模達(dá)到15頭；將AT-Ⅲ純化工藝進(jìn)行逐級(jí)放大，目前能夠達(dá)到30 L/批次，正在嘗試進(jìn)行放大到50 L/批次，蛋白純度達(dá)到98%以上；采用兩種方案對(duì)hFIX蛋白進(jìn)行純化，方案一的比活性和純度高于第二種方案，但方案二的回收率較高；經(jīng)過(guò)對(duì)純化的rhATⅢ和hFⅨ蛋白的結(jié)構(gòu)進(jìn)行確證，均和人源的相似；通過(guò)藥物制劑的研究，rhATⅢ蛋白確定了合適的制劑配方；目前正在進(jìn)行藥品注冊(cè)臨床前研究工作。

關(guān)鍵詞：動(dòng)物乳腺生物反應(yīng)器 抗凝血酶Ⅲ 凝血因子Ⅸ

Abstract：This project is intend to deeply study the mammary gland bio- reactor on the basis of successfully getting the target protein， and comprehensively using molecular biology， cytology， embryology， reproductive science， thremmatology， biochemistry， pharmacology and other subjects to research the efficacy and safety of pharmaceutical proteins produced by animal bioreactor。We will focus on researching the pilot batch quality control、pharmacology and toxicology about target protein，which are required to be studied in preclinical，so the development of the new biological drug produced by Transgenic animal will be an important step forward. For this project，we obtained mammary gland bioreactor of transgenic cattle and goats with high and stable expression。And we have transgenic goats and 15 transgenic cattle； We will enlarge AT-Ⅲ purification proce- ss progressively. At present，the purification scale can reach 30 L/batch， we try to enlarge it to 50 L/batch； protein purity reach more than 98%. we use two solutions to purify the hFIX protein， specific activity and purity of the first solution are higher than the second solution， but the second solution get higher recovery rate；After confirming the structure of proteins，the purified rhAT-Ⅲ and hF-Ⅸ are similar with human sources；We determined a suitable formula of the preparation about rhAT-Ⅲ through researching the pharmaceutical prepara- tion；Now，we are working on pre clinical about drug registration。

Key Words：Mammary Gland Bioreactor；AntithrombinⅢ；Blood Coagulation factorⅨ

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