Preparation And Bioactivity Characterization Of Recombinant Human Soluble Mutant BAFF

B cell activating factor （BAFF）, a ligand belonging to the tumor necrosis factor （TNF）family, plays a critical role in regulating survival and activation of peripheral B cellpopulations and has been associated with autoimmune disease. In BAFF-deficient mice, Bcell development is severely perturbed, whereas BAFF transgenic mice developautoimmune diseases resembling human systemic lupus erythematosus （SLE） andSj gren’s syndrome. The overexpression of BAFF was also found in patients with SLE,rheumatoid arthritis, Sj gren’s syndrome, and B cell malignancies. These data suggest thatBAFF play a critical role in B cell survival and function regulation, and may be atherapeutic target for some human autoimmune diseases. BAFF is known to interact withthree receptors, BCMA, TACI and BAFF-R that have distant similarities with otherreceptors of the TNF family. The study found that a higher level of expression of BAFFreceptor is not only expressed in normal B cells, and B-cell lymphoma and autoimmunediseases such as malignant B cells and abnormal proliferation. Cell-specific expression ofBAFF receptor, suggesting that BAFF and its receptor may be an important potentialtherapeutic targets for B-cell malignant value-added disease and abnormal activation of thedisease.ObjectiveIn view of BAFF and its receptors are expected to become the new target for treatmentof related diseases. Therefor, we based on the nucleotide sequence of the coding the peoplesBAFF amino acids, its cDNA sequence was artificially mutations, we obtained the solublemutant BAFF（smBAFF） protein coding sequence, to construct the correspondingrecombinant plasmid, recombinant human smBAFF （rh-smBAFF） was expresed and purified, and finally the functional activity of the protein was identified.Methods1. rh-smBAFF genetic synthesis and cloning: According to analysis the structure andfunction domain of the BAFF protein, and the codon bias of E. coli, synthesis humansmBAFF gene and insert into pUC57, obtain the recombinant vector pUC57-smBAFF.According to the optimized smBAFF gene sequences, primers were designed, the targetgene fragments were amplified by PCR, and inserted into pMD19-T cloning vector andtransformed into E. coli DH5α. Colony PCR screening and restriction digestion of thepositive clone was identified by colony PCR screening, restriction digestion and DNAsequencing.2. Construction and identification of expression Vector: Extracted the recombinant cloningplasmid pMD19-T/smBAFF, separated smBAFF gene fragment by Nde I and Xho Irestriction enzyme digestion and agarose gel electrophoresis, inserted into expressionvector pQE-T7-2, transformed into E.coli DH5α, the positive recombinant plasmid wasidentified colony PCR.3. Expression and identification of the rh-smBAFF: The recombinant expression plasmaidpQE-T7-2/smBAFF were extracted and transformed into E.coli BL21, the monclonalcolony was picked and cultured to logarithmic phase, and then induced by IPTG. Therecombinant protein was analysised by SDS-PAGE and Western blot.4. Analysis the expression form of the recombinant protein: The bacteria were split bysonication, cell lysate supernatant and inclusion bodies were extracted, the expression formof the recombinant protein was analysised by SDS-PAGE.5. The purity of rh-smBAFF: The expression form of the recombinant protein wasinclusion bodies, inclusion bodies were extracted and dissolved, and then the smBAFF waspurified by Ni2+-NTA affinity chromatography under denaturing conditions.The purity ofinterest protein was analysised by SDS-PAGE. The purified protein was refolded underspecified conditions, and concentrated by ultrafiltration.6. Receptor binding experiments: The capacity of the recombinant protein smBAFFcombined with B lymphocyte was detected by immunofluorescence.7. Cell proliferation experiments: The functional of recombinant protein smBAFF topromote B lymphocyte proliferation and competitive inhibit the function of the nature soluble BAFF was identificated by CCK-8.SPSS17.0statistical software used for statistical analysis.Results1. The construction of rh-smBAFF cloning vectors and strains: The smBAFF gene wascloned, the recombinant cloning plasmid pMD19-T/smBAFF was successfully constructed.Sequence analysis showed that the cloned gene was same as the optimal sequence ofsmBAFF gene.2. The construction of rh-smBAFF cloning vectors and strains: The recombinantexpression plasmid pQE-T7-2/smBAFF was successfully constructed, and transformedinto competent strain BL21, and then induced by ITPG. The line of SDS-PAGE analysisshowed that there was a distinct protein band in the17KDa, which was consistent with thesize of the target protein. The analysis image scanners showed that target protein accountedabout40%of total protein. Western blot showed that recombinant protein can specificalyreact with both the anti-BAFF polyclonal antibody and anti-His-Tag polyclonal antibody,which indicating that the specificity of the rh-smBAFF.3. Isolation and purification of rh-smBAFF: Bacteria lysate supernatant and inclusionbodies were extracted and analyzed by SDS-PAGE, the SDS-PAGE analysis shows thatrecombinant protein located mainly in the inclusion body, clearaged inclusion body underdenaturing conditions. The gel image analysis of the recombinant protein after purified byNi²⁺-NTA affinity chromatography show that the purity of rh-smBAFF recombinantprotein account to more than90%.4. The rh-smBAFF specific combine with B lymphocyte: The result of immunofluorescen-ce showed that the rh-smBAFF can combine with B lymphocyte.5. The inhibition effects of rh-smBAFF against to sBAFF: The analysis of CCK-8showedthat the rh-smBAFF loss the activity of costimulating B cell proliferation; moreover, itcould competitive inhibit the function of the nature soluble BAFF.ConclusionThe successful preparation of rh-smBAFF which possess B cell binding activity but lossthe activity of costimulating B cell proliferation，these may lay a foundation for study ofthe therapy for B cell malignancies and B cell abnormal activity diseases.