Genetic Variation Of PRRSV CH-1a Strain Attenuated Process And Construction Of Full-length Genome CDNA Clone Of PRRSV CH-1R Strain

In the study, four different generation (PRRSV) CH-la strain was selected in sequencing and analyzing full-length genome sequence of porcine reproductive and respiratory syndrome virus strain,and tried constructing the full-length genome cDNA clone of PRRSV CH-1R strain.Through sequence of four different generation of PRRSV CH-1a .we named the four different generationof attenuated process as P39,P71,P148 and P171.Analyzed the sequence and the coding by the molecule biology software, the results indicated that the variations of the Nsp2 and 3' UTR region were reversely obvious, especially the NSP2.This protein was considered to be species-specific.It seems to be more tolerable to mutations and insertions. Many nucleotide mutations were found in this protein.There is a poly (A) tail in the 3'-UTR. A motif, consisting of eight nucleotides immediately upstream of the poly (A), is also different to CH-1a. The motif is highly conservative among arteriviruses, which suggested that the motif might be important for the RdRp recognition and the initiation of the virus replication.Its biology method up for further study.Seven pairs of primers were designed for sequencing the genome of PRRSV CH-1R. Each gene segment was amplified by RT-PCR .A molecular marker (NheI enzyme site) was introduced through gene silencing mutation for distinguishing wild-type virus and rescue virus. Each gene amplified fragments and pBluescript II SK(+) vector were digested by restriction enzyme to construct full-length genome cDNA(pBl-CH-1R)clone of PRRSV CH-1R strain. Checked up pBl-CH-1R seperately restriction enzyme digested and genome sequenced. The results showed that full-length cDNA clone of PRRSV CH-1R strain was constructed successfully. Not only the whole genome sequence were contained﹑But also SP6 promoter sequence and a molecular marker(NheI enzyme site) were contained.Through squence of four different generation PRRSV CH-1a strains attenuated process and construction of full-length genome cDNA clone of PRRSV CH-1R strain will support material for further study of functional genomics, molecular mechanism of pathogenesis and variation of PRRSV and will show application prospects in research of gene-deleted vaccine and marker vaccine.