鼠层粘蛋白α5链E_3、LG_4、LG_5片段的克隆及表达

Cloning and Expression of LG_4、LG_5 and E_3 Fragment of Mouse Laminin alpha 5 Chain

从鼠的肝脏组织中提取总 RNA,采用 RT- PCR获得了鼠层粘蛋白 ( laminin)α5链的 E3 ( L G4 - 5)、L G4 和 L G5等 3个基因片段 ,与 NCBI数据库参考序列相比 ,同源性在 99%以上。将 E3 ( L G4 ,5)、L G4 和 L G5定向克隆到原核高效表达载体 Pet- 2 8a中 ,构建 Pet- 2 8a- E3 、Pet- 2 8a- L G4 、Pet- 2 8a- L G5等 3个原核表达质粒。将表达质粒转化表达受体菌 BL2 1中 ,IPTG诱导表达。收集菌液进行 SDS- PAGE电泳、Western- blotting分析 ,结果显示 ,这 3段基因在原核细胞中成功表达 ,薄层扫描显示表达蛋白占细胞总蛋白的 2 0 %以上。大量提取包涵体 ,纯化后免疫家兔 ,8周后得多克隆抗体 ,间接 EL ISA在抗体稀释到 1∶ 1 2 80

E_3(LG_(4-5)) fragemet、LG_4 and LG_5 module of laminin alpha5 of mouse were amplified by revese transcription from sliver of embryonic mouse. The genes were cloned into prokaryotic expression vector PET-28a directly, getting three recombinant vectors of Pet-28a-E_3,Pet-28a-LG_4 and Pet-28a-LG_5. The constructed plasmids were transformed into E.coil BL21 and induced with IPTG. The expressed proteins were detected through SDS-PAGE and Western blot. The inclusion bodies imune the rabbit for getting polyclonal antibody. Its titer was detected of 1∶(12 800) by indirectly ELISA.