装载戊型肝炎病毒核酸片段的重组MS2噬菌体衣壳的制备及其特性研究

Preparation and Characterization of Recombinant HEV RNA-loaded MS2 Bacteriophage Capsid by Armored RNA Technology

为制备一种可以实际应用于戊型肝炎病毒(Hepatitis E virus,HEV)核酸检测的质控品,即装载戊型肝炎病毒核酸片段的重组MS2噬菌体衣壳(rHEPC)。根据装甲RNA技术原理,设计将MS2噬菌体基因组的5’端部分非编码区、成熟酶蛋白、衣壳蛋白以及部分复制酶起始位点等编码基因与HEV ORF2保守区部分基因序列串联并合成目标基因MS2-HEV。随后将MS2-HEV基因克隆、插入原核表达载体pET-28b中构建pET-28b-MS2/HEV重组质粒,转化大肠杆菌感受态细胞BL21(DE3),经过1mM/L IPTG诱导培养4h后,SDS-PAGE鉴定表达情况;将验证的表达菌体超声破碎、离心、取上清、去除核酸杂质后再经超滤离心纯化、浓缩,电镜分析回收浓缩的rHEPC形态;梯度稀释rHEPC进行稳定性鉴定包括DNase I、RNase A抗性以及保存期三方面的稳定性试验检测。SDS-PAGE分析结果表明重组MS2-HEV基因获得有效表达,重组rHEPC纯度高无杂带;RT-PCR鉴定结果表明设计的HEV保守基因序列成功地被包装到重组rHEPC中。进一步的病毒颗粒稳定性试验结果表明制备的rHEPC能够有效抵抗高浓度的DNase I和RNase A的降解作用,并且在-20℃条件下可以持续保存7个月而无明显降解。上述结果表明制备的rHEPC达到了作为RNA病毒核酸检测阳性质控品的基本要求。

The purpose of this study was to develop an effective control to be applied in hepatitis E virus（HEV）nucleic acid detection.Construction of an MS2/HEV gene was performed based on an ＂Armored RNA technology＂protocol.The gene included a partial MS2 phage genome including the 5＇UTR,the maturation protein,capsid protein and initiation site of the replicase and a partially conserved sequence derived from the HEV ORF2.The target genes were synthesized and amplified by PCR,and the purified target gene products subcloned into the pET-28 bprokaryotic expression vector to obtain the pET-28b-MS2/HEV recombinant plasmid.SDS-PAGE was used for expression analysis in E.coli BL21（DE3）cells harboring the pET-28b-MS2/HEV plasmid.Centrifugal ultrafiltration was adopted for the purification and concentration of recombinant HEV RNA-loaded MS2 Bacteriophage Capsid（rHEPC）.The morphological identification of the particles was subsequently performed by scanning electron microscopy.Stability of the rHEPC particles were evaluated by challenging with different concentrations of DNase I and RNase A,and also evaluated for long-term storage based on RT-PCR verification.SDS-PAGE results showed that the target MS2/HEV gene could express efficiently in recombinant E.coli BL21（DE3）and RT-PCR results revealed that the designed HEV conserved gene sequence was successfully packaged into MS2phage-like or rHEPC particles.Stability evaluation showed that the prepared rHEPC particles exhibited strong resistance to degradation by DNase I and RNase A and long-lasting protection of coated HEV RNA for at least seven months when stored at-20℃.The prepared rHEPC particles in the present study meet the basic requirements to be used as a quality control material for routine HEV nucleic acid detection.