The Expression And Purification Of LIFP Fusion Peptide And Its Gene On Inhibition Of SKOV3Cell

Objective To construct and identify lactoferricin-immunostimulating fusion peptide（LIFP）gene recombinant plasmid, express and isolate the fused protein in E. ColiBL21and obtain highly purity of the fusion peptide. To study the effect of LIFP genein ovarian cancer cell line SKOV3. Methods According to the sequence of bovinelactoferricin and immunostimulating peptide (PGPIPN) in Genbank, LIFP gene wassynthesized with a soft truss arm GGGGS connecting the two peptides. The sequence ofa factor Xa thrombin cleavage site was inserted at LIFP gene5’ end. The LIFP genewas constructed into the expression vector, pGEX-KG, which then was transformed intoE. Coli BL21.The resulting vector was detected by PCR and sequencing. The cultureconditions were optimized, GST-LIFP was expressed in E. Coli BL21. Then bacteriaexpressing GST-LIFP were pelleted by centrifugation and disrupted by sonication，then the expression of fusion protein (LIFP-GST) were observed by SDS-PAGE，thenLIFP-GST was purified with GST affinity chromatography in AKTA chromatographysystem, and identified by Western blot. The LIFP after Factor Xa digestion wasdetermined by high perform-ance liquid chromatography (HPLC) and the amino acidsequence of LIFP was assayed by Mass spectrometry (MS). The LIFP gene was clonedinto lentiviral expression vector pLJM1, which contained CMV promoter and greenfluorescent protein(GFP)．The recombinant vector pLJM1-LIFP confirmed by PCRand sequencing was cotransfected with the packaging plasmids into293T cells byLipofectamine2000．Virus in the supernatant was collected and the virus titer wasmeasured. The constructed pLJM1-LIFP was used to infect SKOV3and the inhibitionof cell proliferation was determined by MTT in vitro. Cell nucleus changes were assessed by Hoechst33258staining. RESULT The recommented vector wassuccessfully constructed. GST-LIFP was highly expressed in E. Coli BL21, then waspurified and identified. LIFP was prepared and identified by HPLC and MS. Thelentivirus vector targeting LIFP gene has been successfully constructed. Aftertransfection, a large number of293T cells with green fluorescence was observed byfluorescent microscopy. The titer of concentrated virus was1.2×108TU/ML. Afterinfection of the recombinant lentiviurs in SKOV3, LIFP inhibited cell growth andproliferation (P<0.05), and induced cell apoptosis. Conclusion The LIFP fusion peptidewas highly expressed and purified. The LIFP gene inhibited SKOV3cell proliferationand induced their apoptosis in vitro.