摘要
目的 构建耐RNase酶内含中东呼吸综合征冠状病毒（middle east respiratory syndrome coronavirus,MERS-CoV）部分N基因的病毒样颗粒（virus-like particles,VLPs）.方法 将MS2噬菌体包膜蛋白和成熟酶蛋白基因编码序列插入到表达载体pET32a,构建成pET32MS中间载体,再将MERS-CoV N基因和beta-actin基因片段连接到成熟酶蛋白基因的下游,获得的重组载体p32MS-EMC-Beta转化到E.coli BL21 （DE）感受态细胞中进行诱导表达,表达产物进行纯化,然后进行耐酶实验及稳定性实验.结果 获得含MERS-CoV N基因片段的颗粒,可抵抗RNase降解,在37℃保存30d.结论 成功构建了含MERS-CoV N基因VLPs且稳定良好,可作为MERS-CoV的标准品和质控品。
Objective To construct and express ribonuclease-resistant virus-like particles containing the RNA fragmengts of MERS-CoV N gene.Methods The coat protein and maturase gene of E.coli bacteriophage MS2 was amplified by PCR,then the gene was cloned into pET32a to construct the intermediate vector pET32MS.The gene fragments harboring MERS-CoV N gene and beta-actin was cloned into the downstream of pET32MS to construct the prokaryotic expression vector p32MS-EMC-Beta.The recombinant plasmid p32MS-EMC-Beta was transformed into E.coli BL21 （DE） competent cells and induced with IPTG.The virus-like particles were obtained after purification.RNase digestion test and stability test were carried out to observe the stability of the particles.Results The RNase-resistant virus-like particles which was able to express the gene fragments containing MERS-CoV N gene and beta-actin were successfully produced and were shown to be stored stable for 30 days at 37℃.Conclusion The virus-like particles with high safety and stability can be used as positive standards and quality controls in the application of MERS-CoV detection.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2015年第3期259-262,共4页
Chinese Journal of Experimental and Clinical Virology
基金
传染病重大专项课题（2013ZX10004-001,2012ZX10004-215,2013ZX10004-202,2013ZX10004804-007）
国家自然科学基金（81101747）
