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人乳铁蛋白基因克隆及细胞表达研究
引用本文:曹阳,高华颖,于黎,袁晓东,李庆伟,汤敏谦,安利佳.人乳铁蛋白基因克隆及细胞表达研究[J].遗传,2002,24(1):9-14.
作者姓名:曹阳  高华颖  于黎  袁晓东  李庆伟  汤敏谦  安利佳
作者单位:1.大连理工大学化工学院生物工程系,辽宁,大连 116012; 2.辽宁师范大学生命科学院,辽宁,大连 116029; 3.宝生物工程(大连)有限公司,辽宁,大连 116600 1.Department of Bioengineering,Dalian University of Technology,Dalian,116012 China; 2.College of Life Science,Liaoning Normal University,Dalian,116029 China; 3.TaKaRa Biotechnology (Dalian)co.,Ltd,Dalian 116600 China
基金项目:辽宁省优秀青年科研人才培养基金资助项目 ( 9730 14 ),大连市科委复合型人才基金资助项目 (99-2 )
摘    要:通过PCR法直接克隆了2.366kb的人乳铁蛋白基因cDNA序列及800bp的山羊β乳球蛋白基因5′ 端调控序列,并连接到表达载体pLNCX中。利用脂质体包裹含人乳铁蛋白基因cDNA的重组质粒pLNCXHLF,并导入到小鼠乳腺癌细胞株MA∕782中,G418及PCR筛选获得阳性单克隆细胞,增殖后,转染细胞利用海藻酸钠固定化包埋培养,经激素诱导,培养液上清通过Western印记检测证明,转染细胞表达并分泌出人乳铁蛋白,分子质量为34kDa;ELISA法测出,每升培养基(含105个细胞)重组蛋白最高表达量为65mg;抗菌实验表明,所获得的重组人乳铁蛋白具有抑制大肠杆菌生长的作用,而且比人乳铁蛋白标准品作用更强。 Abstract:In this paper,we directly cloned 2.366Kb cDNA sequence of human lactoferrin gene and 800bp 5′flank regulatory sequence of β/lactoglobulin gene from goat by PCR,then connected them with the expression vector pLNCX.The recombinant plasmid pLNCXHLF containing human lactoferrin gene cDNA was transfected into mice mammary tumor cell line MA/782 after liposome transinfection.Positive single clone cells were selected with G418 and by PCR.After proliferating,the transfected cells immobilized and cultured in soldium alginate were induced by hormone.The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLF protein,whose MW is 34 KD.The highest amount detected by ELISA reached 65mg/l medium/105 cells.The result of antibacterial experiment indicates that the recombinant hLF protein has the effect of inhibiting E.coli proliferation;moreover,its activity is superior to the commercial available hLF′s.

关 键 词:脂质体  人乳铁蛋白  固定化  细胞表达  Key  words  human  lactoferritin  
文章编号:0253-9772(2002)01-0009-06

Studies of the Clone and Cell Expression of Human Lactoferritin Gene
CAO Yang ,GAO Hua-ying ,YU Li ,YUAN Xiao-dong ,LI Qing-wei ,TANG Min-qian ,AN Li-jia.Studies of the Clone and Cell Expression of Human Lactoferritin Gene[J].Hereditas,2002,24(1):9-14.
Authors:CAO Yang  GAO Hua-ying  YU Li  YUAN Xiao-dong  LI Qing-wei  TANG Min-qian  AN Li-jia
Institution:Department of Bioengineering, Dalian University of Technology, Dalian, 116012 China. chpfkcy@163.net
Abstract:In this paper,we directly cloned 2.366 Kb cDNA sequence of human lactoferrin gene and 800 bp 5'flank regulatory sequence of beta/lactoglobulin gene from goat by PCR,then connected them with the expression vector pLNCX. The recombinant plasmid pLNCXHLF containing human lactoferrin gene cDNA was transfected into mice mammary tumor cell line MA/782 after liposome transinfection. Positive single clone cells were selected with G418 and by PCR. After proliferating,the transfected cells immobilized and cultured in sodium alginate were induced by hormone. The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLF protein, whose MW is 34 KD. The highest amount detected by ELISA reached 65 mg/l medium/10(5) cells. The result of antibacterial experiment indicates that the recombinant hLF protein has the effect of inhibiting E.coli proliferation;moreover,its activity is superior to the commercial available hLF's.
Keywords:human lactoferritin  liposome  immobilization  cell expression
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