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 isopropylthiogalactoside(IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Westernblot 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 33.2×103 and 30.3×103 respectively. The results of Westernblot 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.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment ofNEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.
ObjectiveTo explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 (NEP1-40) gene and neurotrophin 3 (NT-3) gene into neural stem cells (NSCs).MethodsNSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups: no-load lentiviral vector transducted NSCs (group A), NEP1-40 transducted NSCs (group B), NT-3 transducted NSCs (group C), NEP1-40 and NT-3 corporately transducted NSCs (group D), and blank control (group E). Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection (MOI; 5, 10, 15) for different time (24, 48, 72 hours). Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium.ResultsThe optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C (P<0.05), but no significant difference was found between groups B and D, and between groups A and C (P>0.05). The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B (P<0.05), but no significant difference was found between groups A and B, and between groups C and D (P>0.05).ConclusionNEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.