[Cloning of NEP1-40 gene and expression of its protein].
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 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.


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 (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.


CONCLUSIONS
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.
Gong
Fuliang
F
Department of Orthopaedic Surgery, Second Hospital of Xi'an Jiaotong University, Xi'an Shaanxi, 710004, PR China.
Wang
Kunzheng
K
Yu
Pengbo
P
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D009185:Myelin Proteins / Q000235:genetics* / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification*; D010957:Plasmids; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000166:cytology
D018076:DNA, Complementary; D009185:Myelin Proteins; C457761:NEPI-40 protein, rat; D010446:Peptide Fragments