Preparation And Stability Of Hepatitis E Virus-like Particles By Armored RNA Technology

ObjectiveTo develop an effective quality control material which can be practically applied in hepatitis E virus(HEV)nucleic acid detection according to “Armored RNA technology.MethodsPartial MS2 bacteriophage genome including 5 'UTR,mature enzyme,capsid genes and initiation site gene of coding replicase were designed to link partial conserved sequence derived from ORF2/ORF3 of HEV for the recombination of target MS2-HEV gene,the target gene was synthesized and amplified by PCR method,the purified target gene products were then subcloned into p ET-28 b prokaryotic expression vector to construct p ET-28 b-MS2/HEV recombinant plasmid;SDS-PAGE method was used for the expression analysis of identified transformed E.coli BL21(DE3)harboring p ET-28b-MS2/HEV plasmid,and then the centrifugal ultrafiltration method was adopted for the purification and concentration of expressed HEV-like particles and the morphological identification of the particles was subsequently analyzed by electron microscopic scanning;Stability of the HEV-like particles were evaluated by challenging with different concentrations of DNase I and RNase A respectively,and also evaluated by long-term storage in the blood basing on RT-PCR verification.Rusults1 ? PCR identification and sequencing results showed that p ET-28 b-MS2/HEV recombinant plasmid was constructed successfully.SDS-PAGE results showed that the target MS2-HEV gene could express efficiently in recombinant E.coli BL21(DE3)with an expressing 14 k Da band,and the further purified recombinant HEV-like particles obtained high yield with an expected single band in SDS-PAGE.2?Electron microscope scanning result confirmed an approximate 27 nm Virus-like particles existing,RT-PCR verified the appearance of 349 bp of target band in electrophoresis.These results revealed that the designed HEV conservative gene sequence was successfully packaged into MS2 phage capsids and assembled into HEV-like particles.3?Stability evaluation results showed that the prepared HEV-like particles exhibited strong resistance to DNase I and RNase A attack and long-lasting protection of coated HEV RNA at least 6 months under the condition of artificial simulated-20 ? in the blood sample.ConclusionsHEV-like particles was preparated successfully,and the particles possess strong stability.This feature meets the basic requirements of being a quality control material for the routine HEV nucleic acid detection.