人乳铁蛋白cDNA的克隆及在家蚕细胞中的表达

cDNA Cloning of Human Lactoferrin and its Expression in BmN Cells

从人的乳腺组织中克隆了人乳铁蛋白(hLF)cDNA,其DNA序列与GenBank中另外4个hLF cDNA序列具有高度的同源性(同源性达到99%)。将人乳铁蛋白cDNA克隆在质粒pBacPAK8的BamHⅠ位点和XhoⅠ位点,构建成重组转移质粒pBacPAK-hLF。该质粒DNA与已线性化BacPAK6DNA共转染家蚕细胞BmN,在培养的贴壁细胞中挑出空斑,经过3轮纯化,获得重组病毒BmNPV-hLF。蛋白质免疫印迹法检测到在重组病毒感染的家蚕细胞中存在人乳铁蛋白基因的表达产物,分子量约78kD,ELISA法测定结果表明家蚕细胞中重组人乳铁蛋白(rhLF)相对表达量在表达120h达到最高值,约为13.5mg/L。体外生物活性试验表明重组hLF对大肠杆菌JM109具有抑菌活性。

Human lactoferrin cDNA was amplified by RT-PCR from normal human mammary tissue, The comparision with other four hLF cDNA sequences registered in GenBank was shown 99% homology in DNA sequence. The hLF gene was inserted into the baculovirus transfer vector pBacPAKs, and the recombinant plasmid pBacPAK-hLF was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-hLF was purified three times by screening plaques and identified by Southern blotting. The recombinant human lactoferrin （rhLF） was expressed in BmN cells infected with recombinant virus. The expressed products were run on the 7.5% SDSPAGE and their immunoreactivities were determined by Western blotting, The highest expression level of rhLF is about 13.5 mg/L in BmN cells. The bioactivities of the recombinant proteins showed its bacteriostasis effect on E. coil JM109,