| Autor: |
Ying, Liu, Shen, Wang, Tiezhong, Zhou, Shenyang, Gao |
| Rok vydání: |
2018 |
| Předmět: |
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| Zdroj: |
Bing du xue bao = Chinese journal of virology. 32(5) |
| ISSN: |
1000-8721 |
| Popis: |
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 b prokaryotic 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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