Studies On The Inhibition Of TGEV’s Proliferation In PK-15 Cells By ShRna

Porcine transmissible gastroenteritis, characterized by severe diarrhea, vomiting and dehydration, is an acute and highly contagious disease caused by transmissible gastroenteritis virus(TGEV). Pigs of any age and breed can be infected, especially sucking piglets about 2 weeks old, which results in enormous economic loss in swine-producing areas in the world. No conventional medicine has a significant therapeutic effect on TGE and failure of immunization occurs occasionally, although vaccination and slaughter of infected animals may be helpful in controlling the TGE. RNA interference(RNAi) is one of the posttranscriptional gene silencing phenomena, which mediates homologous in sequence-specific m RNA and induces the m RNA degradation with the double strand RNA(ds RNA). RNAi is regarded as an effective strategy for anti-virus infection because of the ability to knock out the target gene.TGEV is a member of the coronaviridae family, which possesses a large 2.86×104 single-stranded sense RNA genome, and is comprised of four structural proteins. Previous research had found that M gene plays a key role during the morphogenesis of TGEV. In this study, based on the sequence of M gene and design principle of shRNA, three pairs of shRNA sequences were designed and synthesized for the construction of RNAi vectors. These recombinant plasmids were transfection into cultured PK-15 cells before inoculation. Then, the features of inhibition of TGEV’s proliferation by shRNAs were analyzed according to the TGEV virus titer and protein expression and the virus genome RNA replication, respectively.The main results of this study are as follows:1. M gene of TGEV CQ strain was amplified from viral RNA for obtaining nucleotide sequences. The results based on bioinformatic analysis showed that TGEV M gene is highly conserved in structure and have closed relationship with SHXB strain(Nanjing). M protein of TGEV has a signal peptide sequence between 16 th and 17 th amino acid residue in N terminal, following by three transmembrane domains. Moreover, it was predicted that the secondary structure of M protein mainly includes β-sheet and β-turn.2. Three siRNAs target to the sequences of TGEV CQ strain M gene at 103-121, 358-376 and 625-643 were designed in accordance with the sequences analysis. The double-stranded DNA coding shRNAs were formed by annealing and inserted into plasmid expressing vectors pRNAT-U6.1/Neo. When reaching a confluence of 50%-70%, Pk15 cells were respectively transfected with different shRNA-expressing plasmids and the transfection efficiency was assessed by the florescence microscope.3. 24 hours after transfection, the cells were infected with 1×103 TCID50 TGEV, and 48 h after infection they were analyzed by cytopathic effect observation and virus titer detection and indirect immunofluorescent assay(IFA) and real-time RT-PCR assay. CPE analysis showed that cells in control group suffer from typical cytopathy. By contrast, pRNAT-2 and pRNAT-3 exhibited ability to relief specific cytopathy caused by TGEV on PK-15 cells. Result of TCID50 and IFA also indicated that at 48 h post-infection, shRNA-treated(pRNAT-2 and pRNAT-3) cells could inhibit the proliferation of TGEV in protein level. Moreover, real-time RT-PCR assay revealed that both three shRNAs have different capable of inhibiting viral replication by 13%,68% and 70%, and shRNA-expressing plasmids completely targeting conserved domain have better performance.In short, siRNA targeting sequences of M gene at 358-376 or 625-643 could protect PK-15 cells from TGEV infection by inhibiting viral replication in levels of m RNA and protein, which can provide the target site for filtering specific molecule inhibitors and provide a theoretical basis for the development of new drugs.