摘要
对SARS病毒核蛋白(N)基因进行了克隆和序列测定。根据GenBank中已发表的SARS全基因组序列,设计合成了1对特异性引物,对SARS病毒N蛋白基因进行了RT-PCR扩增。将PCR产物纯化后与pGEM-T连接得到重组质粒pGEM-N,进行核苷酸序列测定。结果该基因全长1269bp,编码422个氨基酸。与Tor2、Urbani和TW1株相比,核苷酸和推导的氨基酸序列的同源性均为100%。将pGEM-N双酶切,回收目的基因片段并克隆到大肠杆菌表达载体pET28a中,构建了重组质粒pET-N;将其转化表达菌BL21(DE3)用IPTG进行诱导表达。SDS-PAGE结果表明:重组菌可表达相对分子量约为53kD的蛋白。Western-blotting证实,重组N蛋白可以与SARS免疫血清发生特异性反应。经凝胶薄层扫描分析,重组N蛋白表达量约占菌体蛋白的43%。
<Abstrcat> Nucleoprotein(N) gene of SARS coronavirus (SARS-CoV) was successfully cloned and sequenced. The N protein gene was amplified by RT-PCR with a pair of specific primers, then cloned into pGEM-T and sequenced. The results of sequencing showed that the full length of N gene was 1 269 bp and encoded 422 amino acids. The homology of nucleic acids and amino acids were all 100%, respectively, compared with the standard strains Tor2, TW1 and Urbani. Then nucleoprotein gene was subcloned into the prokaryotic expressing vector pET28a. Positive recombinant was transformed into E. coli strain BL21(DE3) for expression under induction of IPTG. The results of SDS-PAGE revealed that the molecular weight of expression product was 53 kD, which could be specifically recognized by polyclonic antibody against SARS virus through Western blotting analysis. The expression of recombinant N protein of SARS could amount to 43% in the total protein of the induced bacteria by gel scanning analysis.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2005年第3期331-334,343,共5页
Journal of Jilin Agricultural University
基金
吉林省教育厅资助项目(20030612)
