关键词: 鸡BF2基因; 胞外区; pMAL/p2X系统; 可溶性表达; 纯化; 切割 

Abstract: Total RNA from our laboratory stored spleens of four Sanhuang-chickens， one Wuji-chicken and one Zhenzhu-chicken were extracted respectively using Trizol reagent (Gibco/BRL). First-strand cDNA (fcDNA) were generated from total RNA using primer P15T: (5’-CTG ATC TAG AGG TAC CGG ATC CTT TTT TTT TTT TTT T-3’). According to the sequence of chicken BF2gene in GenBank， Two pairs of primers containing EcoRⅠ and Hind Ⅲ restriction sites was designed to clone BF2 genes extracellular domains.After being purified and recovered, the PCR product was inserted into the pGEM-T Easy vector. The recombinant pGEM-T Easy/BF2(1-5) plamid was digested by double enzymes (EcoRⅠand Hind Ⅲ ). The DNA fragments of interest were subcloned into into pMAL-p2X expressed vector between the EcoR I and Hind Ⅲ restriction sites (named p2X-BF2(1-5), respectively) and transfected into E. coli TB1. The clones were sequenced on both strands by the Shanghai Sangon Biotechnology Company.The recombinant plasmids p2X-BF2(1-5) were identified by EcoRⅠ and Hind Ⅲ. The engineering TB1 strains containing p2X-BF2(1-5) were induced by IPTG and SDS-PAGE analysis was carried out to identify the expressed protein.The result revealed the BF2 gene extracellular domains were composed of 807 bp-819 bp nucleotides encoding 269-273 amino acid residues. The expressed MBP-eBF2 fusion proteins were purified using the amylose affinity resin column and DEAE-Sepharose ion exchange chromatography, and cleaved by the factor Xa protease. The purified fusion proteins were identified by western blot and create a basis for reconstruct their complex identifying the virus-derived antigenic peptides.

Key words: chicken; BF2 gene; extracellular domains; pMAL/p2X system; gene cloning; soluble expression; purify; cut