Gong Fuliang, Wang Kunzheng, Yu Pengbo
Department of Orthopaedic Surgery, Second Hospital of Xi'an Jiaotong University, Xi'an Shaanxi, 710004, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Jan;20(1):9-12.
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.
The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenile 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 isopropyl thiogalactoside (IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Western-blot test.
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 (BamH I 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 x 10(3) and 30.3 x 10(3) respectively. The results of Western-blot test confirmed that the antigenicity of the two proteins was precise.
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.
从大鼠脊髓中克隆nogo - 66和NEP1 - 40基因，并实现其蛋白的体外表达。
采用RT - PCR技术从幼年大鼠脊髓中克隆nogo - 66和NEP1 - 40基因，通过基因偶联作用将目的基因连接到T载体，对重组质粒进行测序，再将基因克隆到PQE30 - GST载体中，然后用异丙基硫代半乳糖苷（IPTG）诱导重组质粒表达蛋白。两种蛋白经镍柱纯化并用Western - blot检测。
成功从大鼠中克隆出Nogo - 66和NEP1 - 40基因，加上酶切位点后各为215 bp和137 bp。测序后未检测到这两个基因的突变。用两种酶（BamH I和Hind III）对表达质粒进行酶切，电泳结果可见目的条带。用IPTG诱导表达质粒，得到纯化的GST融合蛋白nogo - 66和NEP1 - 40，其相对分子质量分别为33.2×10(3)和30.3×10(3)。Western - blot检测结果证实这两种蛋白的抗原性确切。
利用基因工程可在体外高效表达Nogo - 66和NEP1 - 40蛋白，为进一步研究其功能及脊髓损伤疫苗奠定了良好基础。
