鸡新城疫病毒Lasota株HN蛋白主要抗原表位基因在大肠杆菌中的表达

Expression of the Main Genes Encoding Epitope of HN Protein of NDV Lasota Strain in E.coli

用RT—PCR法从NDV Lasota疫苗株中扩增出HN基因，将其克隆到pGEM—T easy vector中，构建了重组质粒pT—HN．设计了1对引物，用PCR法扩增出HN蛋白主要抗原表位基因（442—1734bp共1239bp），将其连接到原核表达载体pET-28a（＋），构建重组质粒pET—HN．用IPTG诱导使其在大肠杆菌BL21（DE3）中表达．SDS—PAGE结果表明，NDV HN主要抗原表位基因在pET—HN中获得了高效融合表达，其表达蛋白的分子量为28kD．同时，通过试验摸索并确定了表达HN蛋白主要抗原表位基因的最佳诱导条件：IPTG终浓度为0．4mmol·L^-1，诱导时间为4h，温度为37℃．

HN Gene of Newcastle Disease Virus was amplified by RT-PCR from NDV Lasota strain and was cloned into pGEM-T easy vector, thus the recombinant expression plasmid pT-HN was formed. A pair of primers were designed and the main genes encoding epitope of HN gene （442 - 1 734 bp, totally 1 239 bp）was amplified and then it was inserted into pET-28a（ ＋ ）to construct a recombinant plasmid pET-HN. The target protein was expressed in E. coli BL21 （DE3） induced with IPTG. The results of SDS-PAGE indicated that the main genes encoding epitope of HN gene was expressed from pET-HN induced by IPTG at high level,and the expressed fusion protein was 28 kD approximately in molecular mass. The result provided fundamental data and materials for the further investigation on immunogenicity and molecular biological function of HN of NDV. The experiment showed that the expression was optimized with proper inducing conditions of 0.4 mmol·L^-1 IPTG, 4 hours and 37 ℃ induction.