蚓激酶重组逆转录病毒载体pLN-BCP-LK^R的构建及其在奶牛乳腺组织中的表达  

Construction of Recombinant Retroviral Vector Containing Lumbrokinase Gene and its Expression in Cow Milk

作　　者：周兴[1] 张居农[1] 

机构地区：[1]石河子大学动物科学技术学院,新疆石河子832003

出　　处：《草食家畜》2007年第1期17-20,共4页Grass-Feeding Livestock

基　　金：国家"863"高技术研究发展计划(No.2002AA206653)

摘　　要：构建含有蚓激酶基因(Lumbrokinase,LK)cDNA的重组逆转录病毒载体pLN-BCP-LKR,以200μg、500μg、1000μg、2000μg的剂量梯度注入泌乳期黑白花奶牛乳腺,转染泌乳期奶牛乳腺组织获得暂时表达,并以500μg为最佳表达剂量。利用脂质体转染PA317包装细胞,通过G418进行筛选得到了阳性细胞克隆,克隆扩大培养后病毒上清感染NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/ml。再用收获的病毒上清转染妊娠期奶牛乳腺组织,产后奶样经FAPA法检测蚓激酶活性,结果表明牛奶具有明显的纤溶活性,也就是说蚓激酶基因已经整合到奶牛乳腺组织并能实现相对稳定的表达。Recombinant retroviral vector including Lumbrokinase eDNA was constructed. The results of FAPA showed that LK gene had been expressed in the cow milk by injecting the plasmid into the galaetophore of cow, which the dosage gradient was 200μg,500μg,1000μg,2000μg. It indicated that the optimal expression dosage was 500μg. The vector was transfected into PA317 cells for packaging by liposomal transfection, by using G418 selection for 2 weeks, G418 - resistant PA317 colonies were obtained and amplified. The supernatant cell culture was harvested and infected NIH3T3 cells, as were to measure the viral titer of recombinant retrovirus, results showed that the highest titer of viral supernatant was 1×10^4CFU/ml. The supernatant of cell culture was injected into the mammary gland of pregnant cow. The result of FAPA showed that LK gene bad been integrated into the mammary gland and expressed continuously in cow milk.

关 键 词：蚓激酶 重组逆转录病毒 包装 转染 表达 

分 类 号：S852.65[农业科学—基础兽医学]