Construction Of Eukaryotic Expression Vector Of Rat Epidermal Growth Factor Gene And Ciliary Neurotrophic Factor Gene And Their Expression In Cos-7Cell

Objective To construct the eukaryotic expression vectors of pSecTag2/Hygro B-EGFand pSecTag2/Hygro B-CNTF, and detect their expression in cos-7cell so as to provideexperimental evidence for gene therapy on spinal cord injury．Methods①Cloning, sequencing, construction and detection of eurokaryotic expressionvector of rat EGF gene and CNTF gene.A SD rat aged3weeks was selected and killed by dislocation of neck, thesubmandibular gland and sciatic nerve were quickly taken out under cold condition,then dissected the submandibular gland and sciatic nerve, after that, the total RNA ofthe submandibular gland and sciatic nerve was extracted under the condition withoutRNA enzyme contamination with Trizol reagents. Using revers transcription PCR, thecDNA fragments of EGF and CNTF genes were amplified from total RNAs respectively,After the end of electrophoretic analysis, the purified PCR products were collected.Then the amplified fragments were respectively inserted into eukaryotic expressionvector pSecTag2/Hygro B to construct the recombined plasmid that encoded EGF andCNTF cDNA．The plasmids carrying EGF and CNTF genes were transfected alonerespectively into competence JM109E.coli. After preliminary screening by PCR, thetwo positive clone were digested by double restriction enzyms BamHⅠand HindⅢ.Meanwhile two positive clone were sequenced by shanghai songon corporation.②transfecting cos-7cells, expressing and detecting recombinant rat EGF protein and CNTF protein.The purified plasmids carrying EGF and CNTF genes were transfected alonerespectively or cotransfected into cos-7cells by liposome method. After transfecting48hours, collecting cell culture medium and then the expression proteins were detected byWestern blot．Results①Using reverse transcription PCR, rat EGF gene and CNTF gene weresuccessively obtained. Two groups were random selected ten recombinant vector’spositive clones respectively, then using polymerase chain reaction, two positive clonewere screening from two groups respectively. unification of the EGF gene and CNTFgene in the constructed vectors and target sequences in the gene bank were confirmedby DNA sequence anlysis.②Then two recombinant plasmids were transfected alone respectively orcotransfected cos-7cells by liposome reagent.48hours after transfected， therecombined EGF protein and CNTF protein were detected in the specific6kDa and26kDa protein bands．Conclusion We successfully obtained the recombinant plasmids pSecTag2/HygroB-EGF and pSecTag2/Hygro B-CNTF. After cotransfecting cos-7cells, the recombinantEGF protein and CNTF protein were detected．...