摘要
从鸡脾脏中提取总RNA，以总RNA为模板，5＇-ATC TAG AGG TAC CGG ATC C-（T）15—3’为反转录引物，采用TaKaRa AMV反转录酶合成First—strand cDNA（fcDNA）。根据GenBank中登录的鸡的BF2基因胞外区序列，设计了含EcoR Ⅰ和Hind Ⅲ酶切位点的2对引物，分别扩增鸡BF2的胞外区基因。PCR产物经纯化回收后，插入到含LaeZ基因的原核克隆载体pGEM—T Easy中，转化至大肠杆菌JM109感受态细胞，经EcoR Ⅰ和Hind Ⅲ双酶切及PCR鉴定，筛选出阳性克隆。再把所克隆的片段亚克隆到表达载体pMAL-p2X vector，构建重组质粒p2X／BF2（1—5）。将重组表达质粒转化人大肠杆菌TB1感受态细胞，筛选阳性克隆，经IPTG诱导表达后，进行聚丙烯酰胺凝胶电泳（SDS-PAGE）分析。结果表明，本研究克隆了5类鸡BF2的胞外区基因序列，试验结果证明本研究已成功克隆并可溶性表达了5类BF2胞外区蛋白质分子，全长约807～819bp，编码约269～273个氨基酸。采用淀粉树脂柱亲和层析和DEAE凝胶过滤方法对表达产物进行了纯化，并对纯化的表达产物进行了western blot鉴定。本研究为进一步利用BF2的胞外区在体外构建MHC工类分子结合筛选抗原表位肽平台奠定了基础。
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 BF2 gene in GenBank, Two pairs of primers containing EcoR Ⅰ 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 （EcoR Ⅰ and 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 EcoR Ⅰ 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.
出处
《中国畜牧兽医》
CAS
2007年第9期53-57,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金资助项目(30371098)
作者简介
李新生（1969-）,男,河南人,博士,副研究员,主要从事动物分子病毒学研究。
通讯作者：夏春。E-mail：xiachun@call．edu．cn
