三种乳腺特异表达载体表达特性的比较

Comparison of the Expression Properties of Three Mammary Gland-specific Expression Vectors

为了比较不同来源乳腺特异表达载体的表达特性 ,将人溶菌酶 (hLYZ)cDNA分别克隆入自行构建的p2 0 5C3载体和国外引进的pBJ41和pBC1载体。重组载体p2 0 5C3LYZ、pBJLYZ和pBCLYZ分别用 2 5kDaPEI包裹后 ,通过尾静脉途径注射哺乳期小鼠。注射后 72h采集乳样 ,经微球菌溶解实验证明 ,3组小鼠乳汁中hLYZ的表达量分别为 87、69、60mg L ;从每组小鼠的 1 2种组织提取总RNA ,经Dotblotting检测证明 ,三种重组载体除在乳腺中表达外 ,p2 0 5C3LYZ还在小鼠的脾和肠、pBJLYZ在脾、pBCLYZ在脾和肾中具有一定的异位表达 ;在有hLYZmRNA转录的上述脏器中用微球菌溶解试验均可检测到hLYZ活性。这些实验结果表明 ,自行构建的乳腺特异表达载体的表达特性与从国外引进的表达载体无明显差别。

To compare the expression properties of self-constructed mammary gland-specific vector p205C3 with vectors pBJ41 and pBC1 obtained from abroad,hLYZ cDNA was subcloned into the three vectors.After mixing with 25kDa polyethylenimine (PEI),the three recombinant vectors,p205C3LYZ,pBJLYZ and pBCLYZ,were injected into mice in lactation via tail vein route,respectively.Micrococcal lysis assay of the milk samples showed that the cDNA was efficiently expressed in the mammary glands with similar expression levels ranging from 60 to 87mg/L.Different tissues of each vector-injected mouse were assayed for transcription of hLYZ mRNA by dot-blotting and for expression of hLYZ activity by Micrococcal lysis assay.The results showed that expression of the three recombinant vectors was mainly restricted to mammary glands with some degrees of ectopic expression in spleen,intestines and/or kidney.These data indicate that expression properties of the self-constructed mammary gland-specific vector p205C3 were similar to other two vectors obtained from abroad and usable for developing mammary gland bioreactors.