摘要
为了解北京地区流行的人呼吸道合胞病毒（HRSV）N蛋白基因的特征，并用大肠杆菌表达获得N蛋白，从2006年1月至3月收集的首都儿科研究所附属儿童医院急性呼吸道感染患儿标本中分离到7株HRSV毒株（3株A亚型，4株B亚型），分别经RTPCR扩增得到HRSVN蛋白全基因，克隆至pUCm—T载体中并进行测序和分析；从重组质粒pUCm—N9968中经PCR扩增获得N基因，亚克隆人原核表达载体pET30a（＋）中，得到重组表达质粒pET30a—N9968，转化大肠杆菌BL21（DE3），IPTG诱导培养；SDS-PAGE和Westernblot检测目的蛋白的表达和抗原性。经扩增并测序，7株HRSVN蛋白基因全长均为1176bp，编码一个含391个氨基酸的蛋白；北京地区7株RSV分离株的N蛋白基因之间的核苷酸和推导的氨基酸同源性分别在85．4％～99．7％之间和95．4％～100％之间，进一步证明N蛋白基因的高度保守性。经诱导培养产生大量带6个组氨酸标记的重组N蛋白，主要以包涵体形式存在。N蛋白粗提物经Ni^2＋亲和层析获得较理想纯化。Westernblot结果显示，所表达的蛋白能与特异性单克隆抗体和人血清反应。说明本研究构建的重组pET30a-N9968原核表达质粒使N蛋白获得了高效表达，并且具有特异的抗原活性，为今后进一步研究奠定了基础。
To characterize nucleocapsid （N） protein genes of human respiratory syncytial viruses isolated from children in Beijing and express the N genes in E. coil,seven HRSV strains （three subtype A and four subtype B） were isolated from clinical samples of infants and children with acute respiratory infections and visited the Children＇s Hospital affiliated to Capital Institute of Pediatrics in Beijing during the period of Jan. 2006 to Mar. Full length of N genes from seven HRSV strains were amplified by reverse transcription PCR （RT-PCR）. The seven PCR amplicons were sequenced after cloning into pUCm-T and the sequences were compared with the N genes from HRSVs in GenBank. N gene was amplified from recombinant plasmid pUCm-N9968 by PCR and then sub-cloned into the prokaryotic expression vector pET30a（＋） after digestion with EcoR Ⅰ and Xho Ⅰ The pET30a-N9968 was transformed into E. coli 13L21 （DE3） and ex pressed by inducing with IPTG. Target protein was characterized by SDS-PAGE electrophoresis and Western blot. The amplified N genes were 1 176 bp in length and the deduced N proteins were 391 amino acids in length. The nucleotide identities of N genes among these seven strains were 85.4%-99.7% and the deduced amino acid similarities were 95.4%-100%. The recombinant plasmid pET30a-N9968 had correct open reading frame confirmed by dual-enzyme digestion and sequence analysis. The fusion protein 6 ·His N was produced after inducing by 1 mmol/L IPTG at 37℃ A unique protein band with molecular weight 49 kD was characterized by SDS-PAGE and purified by Ni^2＋ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein had specific binding reaction to specific monoclonal antibody and human sera, indicating that the expressed protein is of specific antigenicity.
出处
《病毒学报》
CAS
CSCD
北大核心
2007年第6期459-465,共7页
Chinese Journal of Virology
基金
北京人类疾病基因诊断基础性研究实验室科研项目(JS96004)
北京市科委新星计划(2004B34)资助
关键词
人呼吸道合胞病毒
核(N)蛋白
序列分析
原核表达
抗原性
human respiratory syncytial virus
sequence analysis
nucleocapsid protein
prokaryotic expression
antigenic activity analysis
作者简介
孙宇（1978-），女，山西人，实习研究员，从事呼吸道病毒感染性疾病的病原学研究；
邢江峰（1980-），男，山西人，硕士研究生，从事呼吸道病毒感染性疾病的病原学研究。
通迅作者：钱渊，女，研究员，E-mail：yqianbjc@263．net
