Expression of porcine RANKL protein using insect cells and analysis of its biological activity
The porcine RANKL gene was cloned into the baculovirus donor plasmid pFastBacHTA,and the positive recombinant donor vector pFastbachTA-RANKL was identified by restriction enzyme diges-tion and sequencing.The recombinant vector pFastbachTA-RANKL was transformed into DH10Bac competent cells containing Bacmid to construct baculovirus expression vector.The recombinant baculovirus con-taining porcine RANKL gene were obtained via sf9 insect cells transfected by recombinant transposon Bacmid-RANKL using Liposome.The identification results of SDS-PAGE,Western-blot and IFA showed that RANKL protein was expressed successfully by recombinant baculovirus in sf9 insect cells.IPEC-J2 cells were treated with expressed RANKL protein,and M cell-associated gene were tested by PCR assay.The re-sults showed that the expression of M cell-specific marker genes CK-18,Marcksll,Sgenel,Spib,CCL20,UBD,NCF4,and TRAF6 were significantly up-regulated in cells treated with porcine RANKL,indicating the expressed porcine RANKL protein has biological activity and could induce IPEC-J2 cells differentia-tion towards M cells.In conclusion,the successfully expressed porcine RANKL protein provides the ma-terial basis for further study of M cell differentiation in porcine intestinal tract and development of M cell targeted vaccines.
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