Structure Prediction, Cloning, Prokaryotic Expression And Identification Of Fusion Protein D-EGF

Objective: To recombine the protein d-EGF having speciel functions, prediction tothe secondary structure, tertiary structure of d-EGF was carried out and its effect tobioactivity was analyzed. The d-EGF fusion proteins were expressed by prokaryoticexpression system and the expressed product was analyzed and identified. Methods:The secondary structure of d-EGF fusion protein was predicted by the software ofDNAStar and tertiary structure was predicted by the software of Insight II.β-defensin-3plasmids and EGF plasmids were constructed. Recombinant-PCRmethod was applied to integrate EGF gene sequence and β-defensin-3gene sequence.Fusion gene was cloned into the prokaryotic expression vector pET-SUMO. Targetvector pET-SUMO d-EGF was identified by PCR and sequencing, which was theninserted into E. coli BL21(DE3). Estimation by SDS-PAGE, western-blot was used tovalidate results of IPTG inducing and Ni-trap column purification. MTT assay wasapplied to analyze its activity in promoting cell proliferation and drug sensitivity bydisc diffusion test, microdilution method was applied to detect its antibacterial activity.Results: Results revealed that d-EGF protein contained more β-sheet regions havemore positive charges in3-22,31-48areas. The six-chain disulfide bond inβ-defensin-3, EGF tertiary structure was necessary to their activities. All of them canbe sustained in fusion d-EGF, that is to say, which maintained β-defensin-3tertiarystructure of the three antiparallel β-pleated sheet structure in the fusion protein.β-defensin-3, EGF plasmids were successful constructed. The294bp fragment wassuccessfully amplified by the recombinant PCR. Recombinant PCR results wereconsistent with the expected results and sequencing testified its preciseness. Thenrecombinant plasmids were transfered into E.coli BL21(DE3). The target protein wasinduced through added IPTG. SDS-PAGE analysis was also used to show about28kDprotein bands, and moreover, western-blot was applied to identify the fusion proteincontaining EGF, β-defensin-3epitope, respectively. Measured by MTT assay, fusion protein d-EGF EC50was approximately0.4ng/ml, which was consistent with EGFstandard EC500.08-0.8ng/ml. Drug sensitivity by disc diffusion test for detection ofStaphylococcus Aureus showed obvious inhibition zone. Quantitative results showedinhibitory concentration was25μg/ml and at the same time standard product humanβ-defensin-3showed MIC12.5μg/ml, which showed inhibitory bacterial activity ofthe d-EGF. Conclusion: Fusion protein d-EGF of second, the tertiary structure wassuccessfully predicted and its activity was preliminarily analyzed. Prokaryoticexpression vector pET-SUMO d-EGF was construted succussfully. Lastly, the targetprotein was expressed in the special system and purified fusion protein was acquired.The d-EGF fusion protein having two kinds of bio-activities was identified bypromoting cell proliferation and antibacterial tests which may offer a new bio-drug inclinical therapy.