Cloning And Expression Of The Gene Encoding B Cell Activating Factor Belonging To The TNF Family And The Study Of Biological Function

The total RNA was extracted from the isolated lymphocytes of the bursa of Fabricius.Throughusing the total RNA as the template and according to the reported sequence, the ORF sequence of thechicken B cell activating factor belonging of TNF family (BAFF) was cloned by RT-PCR. TheRT-PCR product was ligated with the pGEM-T plasmid vector, transformed into E.coli DH 5αand thepositive transfomants was sequenced. Compared with the reported BAFF gene sequence, the similarityof the cloned BAFF gene sequence was 99.88%. The molecular weight of the deduced AA sequenceis 31.6 kDa with.pI 8.3 using DNAssist V1.02.The ORF of chicken BAFF was amplified using specific primers designed according to thecloned sequence, ligated with the plasmid vector pMAL-c2X and then transformed into E.coli Rosset(DE3) by CaCl₂ method.The transformants were identified by PCR and endonuclease digestion.Thesequence of positive clones was analyzed, the ORF of BAFF was solubly expressed through theinduction of IPDG and analysed by SDS-PAGE. The result showed that the molecular weight ofrecombinant BAFF was 74kDa. The rate of expreessed fusion protein on total thalline protein is 15%.The biological activity was measured by MTT method and the results indicated that therecombinant chicken BAFF protein significantly improved the proliferation of chicken spleenlymphocytes.Chicken given i.p. injections of purified recombinant protein, it can increase the spleenweight significantly, but recombinant protein did not significantly protect the lymphocytic damagecaused by IBD vaccines.In this paper, the chicken BAFF was firstly cloned,expressed in our country. At the same time,the biological function of exogenous BAFF was studyed. The result showed that the recombinantprotein can improve chicken immune function in some degree.