NEP1-40基因克隆及蛋白的原核表达和纯化

CLONING OF NEP1-40 GENE AND EXPRESSION OF ITS PROTEIN

目的通过基因工程手段从大鼠脊髓中获取Nogo-66、NEP1-40基因并实现其蛋白的体外表达。方法从幼年大鼠的脊髓组织中,通过RT-PCR技术获得Nogo-66、NEP1-40基因,将目的基因通过基因连接反应与T载体连接,重组质粒进行基因序列测序,构建PQE30-GST和目的基因的高表达质粒,异丙基-β-D-硫代半乳糖(isopropy-lthiogalactoside,IPTG)诱导表达,Ni柱纯化,Western-blot法检测纯化的目的蛋白。结果成功获得大鼠Nogo-66、NEP1-40基因,加上两端的酶切位点和保护性碱基,大小分别为215bp和137bp。序列测序显示基因突变率为0,表达质粒构建双酶切(BamH和Hind)可见目的基因的条带,经IPTG诱导表达,获得带GST标签的融合蛋白Nogo-66和NEP1-40,相对分子质量分别为33.2×103和30.3×103。蛋白纯化结果理想,经Western-blot分析证实抗原性正确。结论Nogo-66、NEP1-40蛋白可通过基因工程手段获得体外高效表达,这将对其功能的研究及脊髓损伤疫苗的研究提供基础。

Objective To clone the genes of nogo 66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. Methods The nogo 66 and NEP1-40 genes were cloned from the spinal cord of juvenil rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropyhhiogalactoside（IPTG） to express the proteins. The two proteins were purified by Ni-column and detected by using Western blot test. Results The Nogo 66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme （BamHⅠ and Hind Ⅲ ）, the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo 66 and NEP1-40, which relative molecular weight were 33.2×10^3 and 30.3× 10^3 respectively. The results of Western blot test confirmed that the antigenicity of the two proteins was precise. Conclusion Nogo 66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.