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 clone human bone morphogenetic protein 2 ( BMP-2) gene and construct the gene’s eukaryotic expression vector. Methods The total RNA was extracted from human osteosarcoma cells, the human BMP-2 cDNA was amplified by RT-PCR and inserted into pGEM-T vector. The positive clones were screened out, and the n the recombinant plasmid was confirmed by restriction enzyme digestion, PCR and the analysis of nucleotide sequence. The BMP-2 cDNA in the pGEM-T cloning vec tor was inserted into the pcDNA3.1(+) eukaryotic expression vector. Results The agarose electrophoresis showed that the fragments of BMP-2, pGEMT and pcDNA3.1(+) were 1.2 kbp, 4.0 kbp and 5.0 kbp, respectively. The result of nucleotide sequence confirmed that the cDNA sequence, which was inserted into pGEM-T and pcDNA3.1(+) plasmid was human BMP-2. Conclusion The pcDNA3.1(+)-hBMP-2 eukaryotic vector can be successfully constructed.
Objective To generate eukaryotic expression vector of pcDNA3.1-β-site amyloid precursor protein cleaving enzyme (BACE) and obtain its transient expression in COS-7 cells. Methods A 1.5 kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells by the RT-PCR method and was cloned into the plasmid pcDNA3.1. The vector was identified by the double digestion with restriction enzymes BamHI and XhoI and was sequenced by the Sanger-dideoxy-mediated chain termination. The expression of the BACE gene was detected by immunocytochemistry. Results The results showed that the cDNA fragment included 1.5 kb total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells transfected with the pcDNA3.1BACE plasmid expressed a high level of the BACE protein in the cytoplasm. Conclusion The recombinant plasmid pcDNA3.1-BACE can provide a very useful tool for the research on the cause of Alzheimer’s disease and lay an important foundation for preventing Alzheimer’s disease.
Objective To construct a mammalian expression vector ofbasic fibroblast growth factor (bFGF) and to investigate the expression of bFGFin vitro and in vivo. Methods A mammalian expression vector pcDNA3.1/myc-His(-)C-bFGF was constructed with gene cloning technique. The mammalian expression system was prepared and purified. The expression of bFGF cDNAin cultured transfected cells was examined by RT-PCR and cell immunohistochemistry. The recombinant plasmids, pcDNA3.1/myc-His(-)C-bFGF and pCD2-VEGF121, were transferred into rabbit cervical muscle by direct injection of plasmid following electric pulses in vivo. The transferred gene expression and the biological effect were measured by use of histochemistry and immunohistochemistry analysis. Results The eukaryon expression system pcDNA3.1/myc-His(-)C-bFGF could express the target protein bFGF in vitro. The recombinant plasmids, pcDNA3.1/myc-His(-)C-bFGF and pCD2-VEGF121 were transferred into muscles flap in vivo successfully. The active proteins bFGF and VEGF121were expressed at high levels. Blood vessels increased significantly in the muscles, and blood circulation was improved by local angiogenesis. Conclusion Theeukaryon expression vector of bFGF is constructed and can be expressed successfully in vitro and in vivo. That is a primary preparation for the research on tissue transplantation and tissue engineering with bFGF gene therapy.