Inhibition Of Goose Paramyxovirus (GPMV) Replication By RNA Interference Targeted To NP And L Gene

RNA interference(RNAi) is a new technique developed gradually recent years. It lead functionality double strands RNA (siRNA) into the cell, then emerge silencing complex induced by RNA and cutting targeted homology RNA instructed by antisense of siRNA,thus intercept expression of target gene. RNAi suffered high reconstruction of virus researcher from discovered, and considered to be one of the best modus operandi of antiviral infection.Recent years, many scholars undertake interference research of viral replication by the way of artificial induced RNAi, RNAi was used in such virus reaserch ,for instance , HIV,HBV,AIV,HCV,SARS,FMDV,DFV e.g, and all of above gained welladvancement.APMV contains nine serological type, viz APMV1~9. But APMV-1 only have one member of NDV. Orthodox theory generally considered that fowl, turkey, pheasant had affectivity of NDV, fowl was the most sensitivity host, and waterfowl couldn't natural infected NDV. Although since 1997, a deal of areas of our country had the reports that goose group were infected similar hadrovirus of NDV one after another, caused severe invasion and death of goose group, severity threat development of goose and fowl's cultivation in our country, then provoked intimated attention. The pathogen was belonged to APMV-1 accredited by aetiology and biologic activity, was named GPMV. GPMV has been became to one of the principal blight in contemporary goose cultivation at present. In our country GPMV has extremely serious development and epidemic and fairly economy lose every year. But the clinic therapy methods such as high immunity blood serum and antiviral drug, although gained invariably therapeutic effect they should not treat the primary disease fundamentally, meanwhile they took a moment of trouble back at home such as medicine residue and blood serun pollution and so on.GPMV coding six constitutive protein, for nucleocapsid (NP),phosphoprotein (P),matrix protein (M),fusion protein (F),hemagglutinin-neuramidinase (HN) and large protein (L). NP,L and some kind of corpuscular protein compose the thanscription complex, nucleocapsid protein of transcription complex conjunct with RNA compact coiling neucleocapsid core, to serve as template in transcription and duplication. Nucleocapsid protein of neucleocapisd core is the most principal constitutive protein, defend the RNAof viral genome, But just while NP and L protein simultaneously to possess high transcriptase activity. NP and L protein are the most conservative protein in GPMV, design siRNA targeted to conservative domain of NP and L gene is elementary to templet of GPMV transcription and duplication, directly inhibit production of template, thereby to cause the step of translation can not undertake, inhibiting duplication of GPMV from origin. Simultaneously, the research of inhibiting GPMV replication by RNAi settle the fundament for research of GPMV fenome function and exploitation of antiviral drug.The research according to the sample of NP and L genome sequence of GPMV NA-1 strain, used on-line design tool of Ambion company to the theory preliminary screening, then blast analysis the 354 sequences, retained the sequence which were high homologous with most GPMV genome,finally selected four target sequence into the research of experiment. Designed a sequence which had similarly base composition but no homology with the target sequence as negative control. According to the insertion element request of pSilencer 2.1-U6 neo vector, design the DNA insertion sequences targeted to the sequence above and artificial synthetized. Then renaturated the single strand and joined into the vector between BamHI site and HindIII site, extracted the recombinant plasmid, uesd EcoRI and SmaI to cut the recombinant plasmid, accredit result display a 3613bp Klenow fragment and a 908 bp fragment after enzyme cutting, certificated that exgenous gene had been connected to the vector, sequencing by consensus primer T7, the result indicated insertion element was correct and U6 promoter was completed. Named the correct plasimid as p2.1-NP14,p2.1-NP18,p2.1-L60,p2.1-L145,p2.1-Control, then large-scale preparation and depuration.The second chapter according to NP and L genic sequence of GPMV NA-1 strain in GeneBank, designed and coalesced two pairs of primer, and one pair of primer targeted to beta-actin, used SYBR GreenI method to establish the Real-time PCR detected method tagerted to GPMV. Used the cDNA of NA-1 strain transcript as standard preparation, optimized reaction receive a series of condition, targeted the two gene to establish the standard curve, then analysised melting curve. The result indicated the standard curve targeted to NP gene was y = - 0.3163 x + 8.81, the coefficient correlation was 0.998, amplification efficacy was 107.16%; the standard curve targeted to L gene was y = - 0.3021 x + 8.59, the coefficient correlation was 0.998, amplification efficacy was 100.49%; melting curve analytic result indicated there was no primer dimer and non-specificity product. The method of detected GPMV instituted in expreriment has high specificity,high sensitivity and short detecting cycle, provided PSS to detect RNAi depressant effect of viral replication in cell.According to the description of liposome 2000 transfected the RNAi plasmid to CEF and chick embryo, inoculated virus after 6 hours. 36 hours later overviewed the RNAi status by Real Time RT-PCR,virus titer,indirect immunofluorescence and CPE. Then detected RNAi depressant effect in chick embryo by hemagglutination test. The experimental result displayed the four RNAi plasmid have high inhibit effective, the inhibit effective of the plasmid targeting NP gene can achieve to 96.5%, the inhibit effective of the plasmid targeting L gene can achieve to 91.6%.