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    • 5. 发明授权
    • 신규한 플라스미드 pEAT8과 이를 이용하여 알파원앤티트립신 단백질을 제조하는 방법
    • 新型PLASMID PEAT 8及其制备方法
    • KR1019930012114B1
    • 1993-12-24
    • KR1019910010937
    • 1991-06-28
    • 한국과학기술연구원
    • 유명희이승철신화수이기녕이상철
    • C12N15/57
    • A genomic gene encoding of human alpha-1-antitrypsin was cloned into a plasmid pET8C. E.coli BL21 (DE3) was transformed by the recombinant plasmids and a transformant (KCTC 0009BP) carrying the recombinant plasmid pEAT8 was screened. The transformant was cultured in a M9ZB medium at 37 deg.C and IPTG (0.04 mM) was added to the culture at OD600 = 0.7. After incubation at 40 deg.C for 3 hrs, the cell pellet was recovered by centrifugation. The cell pellet was treated by sonication and the precipitate containing alpha-1- antitrypsin was recovered by centrifugation. The precipitate was dissolved in a buffer containing 8M area and the alpha-1-antitrypsin was purified by DEAE-Sephacel chromatography and Mono-Q chromatography of FPLC.
    • 将编码人α-1-抗胰蛋白酶的基因组基因克隆到质粒pET8C中。 通过重组质粒转化大肠杆菌BL21(DE3),筛选携带重组质粒pEAT8的转化体(KCTC 0009BP)。 将转化体在37℃的M9ZB培养基中培养,并将ODT = 0.7的IPTG(0.04mM)加入培养物中。 在40℃温育3小时后,通过离心回收细胞沉淀。 通过超声处理细胞沉淀,并通过离心回收含有α-1-抗胰蛋白酶的沉淀物。 将沉淀物溶解在含有8M面积的缓冲液中,通过DEAE- Sephacel层析和FPLC的Mono-Q色谱纯化α-1-抗胰蛋白酶。