Expression And Functional Study Of Porcine Interleukin-21(IL-21)and Generation Of Monoclonal Antibodies Against Porcine IL-21

Interleukin-21(IL-21),a cytokine with broad immune regulation function,plays crucial roles in both innate response and adaptive response.Currently,researches on human and murine IL-21 are quite comprehensive.However,there are few studies about porcine IL-21.In order to study the functions and mechanisms of porcine IL-21,the recombinant plasmid of pcDNA3.1/IL-21-Fc was constructed and subsequently transfected into 293 T cells for transient expression.The porcine IL-21-Fc fusion protein from cell supernatant was purified using protein A affinity chromatography,the purity and activity of purified porcine IL-21-Fc were determined by Western blot and ELISA,respectively.The biological function of the porcine IL-21 was also explored preliminarily.The results demonstrated that the porcine IL-21-Fc fusion protein was successfully expressed in 293 T cell supernatant,purified porcine IL-21-Fc was a protein with 44 kDa.Further biological function exploration of porcine IL-21 showed that porcine IL-21 combined with CD40 L can stimulate the porcine peripheral blood lymphocytes(PBMC)to secret IgG in a dose-dependent manner,in addition,porcine IL-21 could up-regulate the expression of IL-22 in porcine PBMC.Monoclonal antibody(mAb)is an important tool in protein's functional studies.At present,studies of porcine IL-21 are limited due to the reason of no commercial anti-porcine IL-21 mAbs available.In present study,we successfully isolated 5 hybridoma cell clones stably and specifically secreting mAb against porcine IL-21 from immunized mice with prokaryotic-derived purified IL-21-His.The mAbs' Variable genes fragments were amplified separately by polymerase chain reaction(PCR)from the positive hybridoma cells and sequenced.The homology analysis of the V(D)J gene fragments showed that the 5 mAbs derive from 4 different B cell clones;all of 5 mAb can recognize recombinant porcine IL-21 determined by Western blot and IFA identification;notably,one of the Biotin-mAbs,Botin-1D1,can intracellularly stain IL-21+ T cells analyzed by flow cytometry,which lays the foundation for further sorting and studying porcine IL-21+ T cells such as Tfh cells.In this study,we successfully expressed the porcine IL-21-Fc fusion protein with biological activity by mammalian cell expression system,demonstrating that the porcine IL-21 together with CD40 L could stimulate IgG secretion and porcine IL-21 individually induce IL-22 production in porcine PBMC.In addition,we generated and identified 5 mAbs against porcine IL-21.Taken together,the expression of porcine IL-21 and the generation of mAbs against porcine IL-21 lay a firm foundation for further researches of porcine IL-21.