Efficient Prokaryotic Expression, Purification And Functional Identification Of Human HAPRIL-Fc And HBAFF-Fc Fusion Proteins

A proliferation-inducing ligand(APRIL)and B cell activating factor(BAFF)are two closely related novel members of the tumor necrosis factor(TNF)family.They can promote the proliferation and differation of B lymphocyte and T lymphocyte,they also have a crucial role in both humoral and cellular inununity.lots of experiments has proved that some recombinant cytokines(such as IFN-?.CD40L)as immunoadjuvants with vaccines can obviously improve the effectiveness of vaccine,and could improve animal diseases on immunosuppressive resistance.It can also surmount the side-effects of traditional medicines(antibiotics and chemical drugs).At abroad,the recombinant soluble BAFF(sBAFF)has already been approved as an orphan drug to enter Phase I clinical trial studies for the treatment of patients with common variable immunodeficiency(CVID)and immunoglobulin-A(IgA)deficiency but the domestic research is still blank.Besides,APRIL as an immunoadjuvants has not reported both at home and abroad.So in our study,we have constructed two recombinant plasmids:pET28a-hAPRIL-Fc and pET28a-hBAFF-Fc.Then we transformed the two recombinant plasmids into E.coli BL21(DE3)and obtained two soluble proteins:hAPRIL-Fc(42.9KDa)and hBAFF-Fc(43.6KDa).At last,to assay the immunological functions of hAPRIL-Fc and hBAFF-Fc,flow cytometry,laser scanning confocal microscopy and MTT were employed.In addition,an Open reading frame(ORF)of xlLYZ gene has been cloned and identified from African clawed frog by RT-PCR technology in this study.The xILYZ comprises 462 bp and encoding a polypeptide of 153 amino acids.By analysis of its cDNA sequence and amino acids,xlLYZ containing a putative signal peptide,eight conserved cysteine residues(Cys25,Cys49,Cys84,Cys96,Cys100,Cysl 14,Cys135 and Cys147).The result of amino acid sequences comparison which was performed by using the Clustal W software suggested that Xenopus laevis lysozyme,comparing with other known lysozyme proteins,the sequence identity was fairly high at the amino acid level.Recombinant pET28a-xlLYZ was efficiently expressed and inclusion body was formed.Then protein with bioactivity was got through delution refolding.The obtained protein was analyzed by SDS-PAGE and confirmed by Western blot analysis.The activity of the recombinant lysozyme was exammed by using inhibition zone.The optimum temperature and the optimum pH of the recombinant lysozyme were found to be at 40 ? and at Ph5.5.