Expression,Purification And Biological Activity Analysis Of Porcine RANKL

Nuclear factor-kappa B receptor activating factor ligand(RANKL)is one of the essential members of the tumor necrosis factor family.In addition to playing a vital role in bone immunology,it is increasingly important in mucosal immunity,especially in regulating the differentiation of M cells.Highly concerned by scientists,M cells are the critical gateway for intestinal mucosal immunity.Based on this,this study expressed porcine RNAKL in vitro.Its biological function was analyzed after purification,to provide a scientific basis and material basis for improving the immune response of pig intestinal mucosa.To obtain the porcine RANKL protein in good biological activity,an insect/baculovirus eukaryotic expression system was employed.First,the pig RANKL gene was cloned into pFastBacHTA vector,after PCR identification,double enzyme verification,and sequence determination,the correct recombinant pFastBacHTA-RANKL recombinant plasmid was finally obtained.Subsequently,the constructed pFastBacHTA-RANKL recombinant donor plasmid was transformed into DH10 BAC high-efficiency competent cells.After two rounds of blue and white spot screening,white colonies were selected for expansion culture and then identified by PCR.The results showed that this experiment successfully constructed RANKL recombinant bacmids.After transfecting the recombinant Bacmid into sf9 insect cells,observe the cytopathy,harvest the cells,and lyse them to verify the target protein's expression by SDSPAGE,Western blotting,and IFA.The results show that the recombinant baculovirus can correctly express the porcine RANKL protein.Then,the sf9 cells were lysed by ultrasound,and the supernatant of the ultrasound was purified by nickel column.After washing with different gradients of imidazole solution,the RANKL protein was eluted with 250 mM imidazole and identified by SDS-PAGE and Westernblotting.The results demonstrated that the size of the target protein is 40 kDa.The purified pig RANKL protein was used to treat pig small intestinal epithelial cells(IPEC-J2),and mouse intestinal 3D organoids,respectively,and the expression status of M cell-associated marker genes in the treated cells was detected by qPCR.The results showed that the expression levels of some M cellspecific marker genes CK-18,Marcksl1,Sgene1,Gp2,RANK,SpiB,CCL20,UBD,NCF4,TRAF6 were significantly upregulated compared to the negative control group.It indicates that the porcine RANKL expressed and purified in this study has good biological activity and can successfully induce the differentiation of IPEC-J2 cells and 3D organoids of mouse intestine into M cells.This study's results have laid a good material foundation for further research on the differentiation of pig intestinal M cells and provided a scientific basis for the development of M cell-targeted vaccines that enhance the immune response of pig intestinal mucosa.