摘要
用RT-PCR扩增新城疫病毒(NDV)hn基因,得到大小约1700bp的片段,将其克隆至pGEM-Teasy载体并测定其核苷酸序列。根据测序结果,另设计1对引物带有EcoR限制位点,用PCR扩增基因主要功能区即编码HN蛋白球状头部的hngd片段,将其分别克隆至pSOC和pR质粒soc基因3′端,重组质粒pSOC-HNGD转化E.coliBL-21(DE3)感受态细胞,以终浓度1mmol/L的IPTG诱导表达,在SDS-PAGE凝胶上检测到相对分子质量大小约67000的目的蛋白条带。随后将重组质粒pR-HNGD转化T4宿主菌E2感受态细胞,阳性克隆用溶菌酶缺陷的T4-Z1感染,用不加溶菌酶的SC平板筛选阳性克隆。经PCR鉴定,hngd片段已整合在T4-Z1的基因组中。纯化后的重组噬菌体T4-Z1-HNGD用SDS-PAGE和Western-blot可检测到67000的特异条带。结果表明,HNGD蛋白成功展示在T4噬菌体衣壳表面,且与NDV抗血清具有良好的反应性。
The HN gene with length of 1 713 bp of Newcastle disease virus was amplified by RT-PCR.The PCR product was purified and cloned into pGEM-T easy vector and the recombinant plasmid was sequenced.According to HN gene sequencing result,another pair of primer containing EcoRⅠ restriction site was designed to amplify the HNGD fragment coding for the globular domain,the main functional region of HN protein.Then the hngd fragment was inserted at the 3′ terminus of SOC gene of pSOC and pR plasmid respectively.The recombinant plasmid pSOC-HNGD was conducted to E.coli BL-21(DE3) to induce HNGD protein expression with 1 mmol/L IPTG.A SOC-HNGD fusion protein band with molecular weight of 67 000 was dectected on the SDS-PAGE gel.The recombinant plasmid pR-HNGD was subsequently transformed into E.coli E2 competent cells,a host E.coli.Strain of T4 bacteriophage.The recombinant E2 bacteria was infected by lysozyme defective phage T4-Z1 and hngd fragment was integrated into the T4-Z1 genesome and the positive clone can be screened on the SC plate without lysozyme.The recombinant T4-Z1-HNGD was tested by PCR to confirm the hngd integrated into the phage genesome.The purified T4-Z1-HNGD was detected by SDS-PAGE and western-bloting,the specific band with molecular weight of 67 000 be detected.These results showed that HNGD protein has been successfully diaplayed on the T4 bacteriophage capsid suface and the expression products can specifically reacted with antisera against Newcastle disease virus.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2005年第4期346-349,共4页
Chinese Journal of Veterinary Science
基金
国家"863"计划资助项目(2002AA245051)
关键词
新城疫病毒
T4噬菌体
HN蛋白
展示
Newcastle disease virus
T4 bacteriophage
HN protein
display
