Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology

Construction and Identification of Recombinant Lentivirus Expressing shRNA Targeting to ORF1 and ORF7 of Porcine Reproductive and Respiratory Syndrome Virus Genomes

Author LiZhiJun
Tutor JiangPing
School Nanjing Agricultural College
Course Preventive Veterinary Medicine
Keywords PRRSV lentivirus RNAi cell
CLC S852.65
Type Master's thesis
Year 2011
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Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases of swine industry worldwide, which is caused by porcine reproductive and respiratory syndrome virus (PRRSV). The most prominent advantage of RNAi is a powerful inhibition effect of gene expression and a high degree of sequence specificity. Lentivirus can efficiently infect quiescent and division of cells, which makes it an ideal tool for long-term gene expression. In this study, lentivirus and RNAi technology were combined in order to provide new ideas for further suppressing PRRSV infection. The main contents of the research were as following:1. The H1-shRNA cassette of PRRSV were amplified by PCR and cloned into the lentiviral-vector pHAGE-CMV-MCS-IZsGreen on the basis of previous work in the lab, the correct recombinant lentiviral-vector plasmid and packaging plasmid, coated plasmid were co-transfected into 293FT cells, then the recombinant lentiviruses expressing shRNA targeting PRRSV S1 strain ORF1b, ORF7 were produced, named as rLV-P3 and rLV-N3, respectively. Viral titers were approximately 1.0×107efu/mL.2. Five pairs of small hairpin oligonucleotide targeting ORF1 of PRRSV SY0608 strain were designed and synthesized; the oligonucleotides were annealed to produce complementary double-strand as siRNA gene and then cloned into the lentiviral-vector pLVX-shRNA. The correct recombinant lentiviral-vector plasmid and mixer were co-transfected into 293FT cells, then recombinant lentiviruses expressing the shRNA were produced, named as rLV-NP1, rLV-NP2, rLV-NP7, rLV-NP9 and rLV-NP11 respectively. Viral titers were approximately 5×105 IFU/mL.3. Marc-145 cells were infected with a certain dose of rLV-NP9 and cultured 48h, and then cells expressing green fluorescent protein were isolated sterily through FACS. Morphology of the isolated cells was stable and could be normal passage after expansion. Cell genome DNA were extracted and identified by PCR, the results suggested the correct insertion of shRNA expression cassette into cell genome DNA. And the established Marc-145 cell line was named as shRNAEGFP-Marc-145.4. shRNAEGFP-Marc-145 and Marc-145 cells were traduced with a certain dose of PRRSV SY0608 strain and cultured for 48h. CPE were first observed, virus titer were detected by TCID50 and relative mRNA level of cell supernatant were determined by Real-time PCR, respectively. The results showed that there was no significant difference of CPE of the two cells infected by PRRSV SY0608 strain; virus titer and relative mRNA level of cell supernatant were almost same, comparied with that in PRRSV SY0608 infection group. It proved that shRNAEGFP-Marc-145 cells could not significantly inhibit PRRSV replication.

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