| Human respiratory syncytial virus(h RSV)is one of the major pathogens causing severe lower respiratory tract infections in infants and children under 5 years of age,which can lead to death in severe cases.The elderly and immunodeficient populations are also at high risk of RSV infection.Vaccine is the most effective measure to prevent viral diseases,but today there is still no safe and effective RSV vaccine available.The establishment and applications of reverse genetic manipulation techniques have greatly facilitated the study of RNA virus genome structures and functions and viral pathogenesis,and provided technical support for the construction of live genetically engineered vaccines based on viral genome modifications.In order to construct a reverse genetics system for respiratory syncytial virus A2strain,firstly this thesis constructed a Strand-specific fluorescent quantitative RT-PCR assay to detect RSV genomicRNA replication and transcription in the reverse genetics system using RSV A2 strain as materials.The method can distinguish and quantify cRNA and vRNA strands.Two strand-specific primers with tags were designed and synthesized for RT-PCR against the F gene of RSV.The primers consisted of the segment of tag1 or tag2 unrelated to the RSV gene connected to a complementary sequence of vRNA or cRNA.The same sequences as the tag portions of the reverse transcription primers were then used as the forward primers and the complementary sequences with the different RNA strands as the reverse primers for fluorescence quantitative PCR detection.The standard curve constructed using standard plasmids showed good linear correlation between 10~1 and 10~7 copies/μL,and the minimum detection limit for both cRNA and vRNA was 10 copies/μL.The method was highly specific,sensitive and reproducible.vRNA copy number was detected after 72 h of RSV infection of Hep-2 cells at about 3.15×10~6 copies/μg and the copy number of cRNA was about 1.09×10~6 copies/μg.The method was able to distinguish and quantify the vRNA strands and cRNA strands,which provided a new method to study the replication and transcription mechanism of RSV.RSV A2 strain was used as the materials,RNA was extracted for RT-PCR,cDNA was used as the template to amplify the genomic fragment by segmentation,and the full-length cDNA of RSV A2 strain was cloned into pAC plasmid vector by using cloning strategies such as enzymatic ligation and In-Fusion cloning,and green fluorescent protein(GFP)was inserted between the leader sequence and NS1 gene.The reporter gene was inserted between the leader sequence and NS1 gene,the T7 promoter was inserted upstream of the recombinant plasmid cDNA,the hammerhead nuclease and T7 terminator were inserted downstream,and the T7 promoter expression system was used to realize the transcription of RSV reverse genome.Eukaryotic expression plasmids pcDNA3.1-N,pcDNA3.1-P,pcDNA3.1-L,and pcDNA3.1-M2-1 for N,P,L,and M2-1 proteins were also constructed to be used as the four auxiliary plasmids necessary for RSV rescue.To evaluate the function of the protein encoded by the helper plasmids,a microgenomic plasmid pAC-T7-GFP containing the GFP reporter gene was constructed.The recombinant reporter plasmid pAC-T7-GFP and four helper plasmids were cotransfected with Hep-2 cells inoculated with poxvirus vTF7-3 to provide T7polymerase.Fluorescent cells expressing GFP could be observed 72 h after transfection,indicating that the helper plasmids were functionally active.Similarly,the RSV genomic full-length cDNA plasmid pAC-T7-RSV/GFP was cotransfected with four helper plasmids in Hep-2 cells inoculated with poxvirus vTF7-3 providing T7polymerase for RSV rescue,and green fluorescence was observed in Hep-2 cells as confirmed by fluorescence microscopy.Fluorescence quantitative RT-PCR assays showed that the presence of RSV vRNA and cRNA could be detected in transfected cells.These works laid the foundation for the study of the genome structures and functions of RSV and the constructions of RSV genetic engineering vaccine. |
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