猪催乳素的真核表达与生物活性验证OACSTPCD

[Objective]Prolactin(PRL)has a wide range of physiological regulatory effects,but its pleiotropic mechanism is still unclear.In order to investigate the pleiotropy of porcine PRL,we obtain porcine PRL eukaryotic recombinant protein and verify its biological activity.[Method]The porcine PRL gene was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-GFP+Puro by molecular cloning technology,and the PRL-lentivirus carrying porcine PRL gene was obtained by lentivirus packaging.CHO-K1 cells were infected by the concentrated PRL-lentivirus solution,and the positive cell line named CHO-K1-PRL,which could secrete recombinant PRL protein,was obtained after purinomycin screening.The recombinant protein was purified by nickel column affinity chromatography,and identified by LC-MS/MS mass spectrometry.The biological activity of recombinant PRL was verified by adding recombinant PRL into HC11 cell culture system in vitro.[Result]The recombinant expression vector pCDH-CMV-6His-PRL-6His-EF1-GFP+Puro carrying porcine PRL gene was successfully constructed.The titer of PRL-lentivirus after concentrating was 9.9×108 TU/mL,and the positive cell line CHO-K1-PRL was obtained after puromycin screening.The recombinant protein with the mass concentration of 50 μg/mL was successfully purified from the supernatant of CHO-K1-PRL cells.The recombinant porcine PRL protein was identified as porcine PRL by LC-MS/MS mass spectrometry,and the coverage rate of recombinant PRL was 94%.The recombinant PRL had the biological activity of promoting the proliferation and casein expression of HC11 cells.[Conclusion]The cell line CHO-K1-PRL constructed in this study can stably express porcine recombinant PRL with biological activity,which lays a foundation for the functional research,production and application of porcine PRL.