Expression Of Conserved Dopaminergic Neurotrophic Factor In Sf9 Cells By Using Bac-to-Bac Expression System

Objective: Use the Bac-to-Bac expression system to express recombinant protein CDNF in insect cells.Methods: Mouse total RNA was isolated using Trizol reagent then first-strand cDNAs were synthesized by reverse transcriptase.Full length (564bp) mouse CDNF was amplified by PCR. The PCR product was cloned into pFastBacHTb vector to create pFastBacHTb-CDNF vector which transformed into E.coli DH5a competent cells. Recombinant plasmid was selected by LB agar plates containing 100ug/ml ampicillin, and was further verified by PCR amplification, restriction analysis and sequencing. The constructed baculovirus transfer vector pFasBacHTb-CDNF was transformed into E.coli DH10 Bac competent cells. Blue/white selection was used to identify colnies containing the recombinant bacmid. Successful transposition to the bacmid will be verifid by PCR analysis using pUC/M13 forward and reverse primer. The recombinant Bacmid-CDNF DNA were transfected into Sf9 insect cells using Cellfectinâ…¡reagent according to the manufacturer's instructions. Infected and uninfected sf9 cells can be distinguished by morphology. Recombinant CDNF protein was prduced in sf9 insect cells after infection with recombinant baculovirus, Expression of recombinant protein was identified by western bolt.Results: Agarose gel electrophoresis after RT-PCR showed a 564bp band which consistent with the anticipation size. Recombinant plsmid pFastBacHTb-CDNF was constructed and successfully identified by double digestion of Xho I and BamHI restriction enzyme or single digestion of BamH I, PCR amplification and sequence. After the pFastbacHTb-CDNF transfer vector was transformed into DH10Bac E.coli, recombinant bacmid-CDNF was successfully verified by PCR amplification using pUC/M13 amplification primers and CDNF primer. Recombinant Bacmid-CDNF was isolated from over night cultures and transfected into insect cells sf9 with CELLFECTIN reagents. Uninfected cells continued to divide and form a confluent monolayer while infected cells stopped dividing and enlarged after 48-72 hours. Viral stock was isolated and storaged. Recombinant CDNF protein was induced to expression in sf9 cells. The molecular weight of the recombinant protein CDNF was approximately 21kD(plus with 3kD 6-His tag).Conclusion: The recombinant protein mCDNF was successfully expressed and purified using Bac-to-Bac expression system.