【摘要】 目的构建蚓激酶重组逆转录病毒载体,并在牛乳腺中稳定表达蚓激酶。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中,获得蚓激酶重组逆转录病毒载体,利用脂质体转染PA317包装细胞,G418筛选阳性细胞克隆,克隆扩大培养后,病毒上清转染妊娠7个月奶牛乳腺小叶组织。产犊后采奶样,FAPA法检测蚓激酶活性,并进行凝胶薄层扫描分析。结果蚓激酶重组逆转录病毒最高滴度约为1×104CFU/ml。奶牛产后奶样中蚓激酶基因在牛乳腺中可连续表达112d,活性相当于30000英国单位/L牛奶,在牛奶中表达量可达180mg/L牛奶。结论已成功构建蚓激酶重组逆转录病毒载体,蚓激酶可在牛乳腺中相对稳定地表达。更多还原

【Abstract】 Objective To construct recombinant retrovirus for stable expression of lumbrokinase in mammary gland of cow.MethodsInsert the expression cassette containing lumbrokinase gene into retrovirus expression vector pLNCL and transfect the constructed recombinant plasmid to PA317 cells in mediation of liposome.Screen positive clones with G418 for further culture,and collect the culture supematant for packaging transfection to the lobules of mammary gland of cows during gestation period.Collect the milk samples of the cows after parturition for gel thin layer scanning and determination of lumbrokinase activity by fibrin agarose plate assay(FAPA).Results The maximum titer of recombinant retrovirus with lumbrokinase gene was 1×104 CFU/ml.Lumbrokinase was continuously expressed in mammary gland of cows for 112 d,and its activity and expression level were 30 000 UK units/L milk and 180 mg/L milk respectively.Conclusion The recombinant retrovirus for expression of lumbrokinase was successfully constructed,and lumbrokinase was stably expressed in mammary gland of cow.更多还原