自身启动子控制的卡那霉素抗性基因在哺乳动物细胞中表达的检测

Detection of kanamycin resistant gene expression in vector-transfected mammalian cells and milk samples of vector-injected cow

为了研究卡那霉素抗性(KanR)基因能否在哺乳动物细胞中表达以及用含相同抗性基因重组质粒防治奶牛乳腺炎的安全性,用PCR从重组质粒p215C3LYZ中扩增得KanR基因,将其克隆入原核表达载体pQE-31,用含卡那霉素(Kan)琼脂平板筛选得KanR重组菌,经IPTG诱导成功表达了预期大小的Kan抗性融合蛋白;用纯化的重组蛋白免疫小鼠,获得了Kan抗性蛋白抗血清,经Western blotting证明免疫血清特异性良好;分别用重组质粒pQE-Kan和p215C3LYZ转染COS-1细胞,在不含抗生素培养液中培养后分别收集细胞上清和细胞裂解物;将转染细胞上清分为添加和不加Kan组,接种Kan敏感菌DH5α大肠杆菌,培养物的OD600检测结果显示,添加组的指示菌生长被抑制,转染细胞上清中无Kan抗性蛋白表达;以Kan抗性蛋白免疫血清进行的Western blotting结果显示,转染细胞内无Kan抗性蛋白表达;将p215C3LYZ注射奶牛乳腺,用脱脂和浓缩奶样进行Western blotting检测,结果显示试验牛乳中也无Kan抗性蛋白表达。这些试验结果提示,来源于原核大肠杆菌的KanR基因启动子在动物细胞中无转录活性,乳腺注射含KanR基因的重组质粒不会表达对奶牛和人体有害的Kan抗性蛋白。

To investigate whether the kanamycin resistant （ Kana ） gene in mammary-specific expression vector p215C3LYZ for treating bovine mastitis could express active protein in mammalian cells under the control of its own promoter, KanR gene was amplified by PCR using mammary-specific expression vector p215C3LYZ as the template and subcloned into prokaryotic expression vector pQE-31. After transformation and cultivation on Kan-eontaining agar plates, Kan-resistant E. coli colonies were obtained and an expected recombinant protein was ex- pressed after IPTG induction. After separation on SDS-PAGE, the protein band was excised and specific antiserum was obtained by immuni- zation of mice for 6 times. The specificity of the antiserum was confirmed using Western blotting. The prokaryotic expression vector pQE-Kan and eukaryotie expression vector p215C3LYZ, both of which contained KanR gene, were transfected into COS-1 cells and the supernatants were collected after cultivation in antibiotic-free medium, DHScL E. coli were inoculated into the supematants in the absence or presence of Kan and the OD600 values were detected after 24h cultivation. The results showed that growth of the indicator bacterium was inhibited sigrlificantly in the presence of Kan, indicating that the Kan resistant protein was not expressed in the supernatants of the vector-transfected cells. Western blotting of the cell lysates confirmed that Kan resistant protein was not expressed in the vector-transfected cells. In addition, no Kan resistant protein was detected in concentrated milk samples from cows after intramammary injection with vector p215C3LYZ. These experimental data demonstrate that the promoter of KanR gene is inactive in mammalian cells, providing additional safety evidence using the p215C3LYZ vector for treating bovine mastitis.