摘要
目的 :克隆编码严重急性呼吸综合征冠状病毒 (SARS CoV)N蛋白的DNA ,构建原核表达质粒pGEX 2T/N ,并诱导表达。方法 :采用RT PCR方法从病毒培养液中获得N基因片段 ,并克隆入T EASY载体中。经PCR、双酶切鉴定后 ,测序。将N蛋白基因序列定向插入原核表达载体pGEX 2T中 ,表达融合蛋白。用表达产物与抗SARS CoV抗体阳性血清做Westernblot。结果 :N蛋白DNA测序的结果与GenBank比对缺失 2 0个bp。GST N融合蛋白以可溶形式表达。Westernblot检测表明 ,其与抗SARS CoV抗体阳性血清的反应呈阳性。结论 :成功地构建原核表达质粒pGEX 2T/N ,并表达GST N融合蛋白 ,为下一步的研究奠定了基础。
AIM: To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV and GST in E.coli. METHODS: The N region gene of SARS-CoV was cloned by RT-PCR. The expression vector was constructed by DNA recombination. The recombinant plasmid was transformed into E.coli BL21(DE3). The expression of the fusion protein was detected by Western blot. RESULTS: (1) As compared with the sequences in GenBank, 20 bp were deleted in DNA sequence of the cloned N protein. (2) The fusion protein GST-N was soluble. Western blot analysis showed that the reaction of GST-N to anti-SARS-CoV sera was positive. CONCLUSION: The pGEX-2T/N has been constructed and expressed in the form of fusion protein GST-N successfully, which lays the foundation for further study of SARS-CoV N protein.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期422-424,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目 (No .30 340 0 1 0 )
北京市自然科学基金资助项目 (No .70 340 50 )
