摘要
目的制备人可溶性增殖诱导配体(soluble a proliferation inducing ligand,sAPRIL)的两种突变体,为寻找APRIL的竞争抑制剂创造条件。方法采用一步反向PCR法,以人APRIL第186位甘氨酸(186G)和187位谷氨酰胺(187Q)残基为突变位点,构建如下两个突变体DNA,即sAPRIL突变体-1(mutant sAPRIL-1,msAPRIL-1)DNA(186G被赖氨酸残基K置换,187Q缺失)和msAPRIL-2 DNA(186G187Q置换为186K187G);测序后将两突变体DNA分别亚克隆于原核表达载体pQE-80L,继而在大肠杆菌DH5α中表达相应突变体蛋白,经SDS-PAGE和Western blot鉴定表达产物,经Ni2+-NTA和Sephadex G-75柱层析纯化相应突变体蛋白。结果一步反向PCR结合DNA测序,得到了与上述设计一致的两个sAPRIL突变体DNA。将两突变体DNA分别亚克隆于pQE-80L后,在大肠杆菌DH5α中成功表达了相应突变体蛋白。经Ni2+-NTA柱层析成功地纯化了两突变体蛋白,经Sephadex G-75柱层析成功获得了两突变体蛋白的三聚体分子。结论成功制备了sAPRIL的两种突变体蛋白,为寻找基于sA-PRIL突变体的抗肿瘤分子奠定了基础。
Objective To generate two mutants of human soluble APRIL(sAPRIL) for laying a foundation for developing competitive inhibitor of a proliferation inducing ligand(APRIL). Methods Taking 186G and 187Q of human APRIL as mutation sites, one-step opposite orientation PCR was used to construct the two mutant sAPRIL(msAPRIL) DNAs, namely msAPRIL-1 DNA(in which 186G was replaced by K while 187Q was deleted) and msAPRIL-2 DNA(in which 186G187Q was replaced by 186K187G). The two sequenced msAPRIL DNAs were separately subcloned into the prokaryotic expression vector pQE-80L, and the corresponding recombinant msAPRIL proteins were expressed in E. coli DH5α. The expressed msAPRIL proteins were analyzed by SDS-PAGE and Western blotting, and purified by Ni^2+ -NTA and Sephadex G-75 chromatography. Results By using one-step opposite orientation PCR, the two msAPRIL DNAs were cloned, and their sequences were consistent with the designed mutation, After inserting every msAPRIL DNA into pQE-80L vector respectively, the recombinant msAPRIL proteins were successfully expressed in E. coli DH5α. The msAPRIL proteins were purified effectively; the homotrimers of msAPRILs were isolated by Sephadex G-75. Conclusion Two msAPRIL proteins are successfully prepared, which might pave a way for developing novel antitumor therapeutics based on mutant sAPRIL.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第6期636-639,644,共5页
Immunological Journal
基金
重庆市科委科学基金计划资助项目(CSTC
2006BB5110)
第三军医大学科研基金资助项目(SG200538)
关键词
增殖诱导配体
突变体
原核表达
肿瘤
A proliferation inducing ligand
Mutant
Prokaryotic expression
Tumor