Site-specific Mutagenesis And Identification Of F Gene Of NA-1 Strain Of Goose Paramyxovirus

Avian paramyxovirus consisted of nine serotypes, APMV1~9, but APMV-1 only included Newcastle disease virus. There are tremendous virulence diversity among different hosts in which chicken was the most sensitive, pigeon and parrot also cold be infected and falled ill. Waterfowls such as geese or ducks constantly were not considered to be infected by NDV. For the past few years, uninterruptly expanding of the host infection spectrum about NDV, chicken, pipeon, parrot and some rare birds could be infected,as well as waterfowls. The outbreak of goose-host paramyxovirus disease broken traditionary theory which said waterfowls could not be infected by NDV. The results of our scientific research and people's practice show that it is very difficult to get complete immuno-protection from goose paramyxovirus disease by traditional NDV virulent strain vaccine. It is imperative to construct an immunization protocol that is similar to the immunization protocol of chicken NDV disease in order to prevent the spread of goose paramyxovirus disease.F protein of paramyxovirus is the main agent which determines the virulence of virus. It is the main protective antigen of the virus, and it has very good immunogenicity. Inactive F0 was synthesized by F gene at the first stage; it plays a role of fusion after it was hydrolysis to be F1 and F2 later. Before the filial virus has the capability of infectivity, it is essential that the clearage of F0 to be F1 and F2. The strain that has different toxicity has different F gene. The main difference is that the clearage site of F gene. The amino acid sequence of clearage site of velogenic strain is 112Arg-Arg-Gln-Arg/Lys-Arg-Phe117, and it is 112Arg-Arg-Gln-Gly-Arg- Leu117 in low virulent strain. The amino acids in this area are some basic amino acids in the velogenic strain, but there are some neutral amino acids in the low virulent strain instead, especially the arginine in 112 and 115 are substituted. The amino acid in 117 is the main factors that influence the clearage, it is phenylalanine in velegenic strain, and it is leucine in low virulent strain. Phenylalanine is not essential for clearage, but it inhabit clearage after it was changed to be leucine.NA-1strain of goose paramyxovirus belongs to gene VII NDV. The amino acid sequence of clearage site of F protein is 112Arg-Arg-G1n-Arg/ Lys-Arg-Phe117, and the sequence is the same as the area of virulent strain. According to the international standards of assessment of the virulence of New-castle disease virus, the MDT, ICPI, IVPI are 69.6h, 1.66, 1.65 respectively, and the MLD of goose embryo is 10-7, it is obvious that the NA-1 strain is a virulence strain that has the same characteristic of virulence as NDV. The main purpose of this research is modifying the F gene of NA-1 strain of goose paramyxovirus by site-specific mutagenesis by PCR. The clearage sites from 112 to 115 on the amino acid sequence would have the characteristic of low virulent strain after the F gene was modified. The modified gene is expressed by the Bac-to-Bac expression system. The expression efficiency and immunogenicity were assessed by SDS-PAGE, Western-blot and Indirect immunofluorescence. This research is helpful for construction of low virulent strain of goose paramyxovirus by reverse genetics on the next step in our laboratory.Based on the sequence of F gene of Geese's Paramyxovirus which have been published, Use site-specific mutagenesis by PCR in vitro, two pairs of primers were designed and synthesized. Four new F gene fragments contained the mutational sites of a domestic isolate strain(GPMV NA-1)was amplified buy overlap PCR. Accordingly, the 112 amino acid codon is changed through AGG to GGA, the 115 amino acid codon is changed through AAA to GGA, the 117 amino acid codon is changed through TTT to ATT.The result is that we get a new by mutation, named F'gene.After purified by 1% agarose gel electrophoresis, The F'gene were cloned into the vector of PGEM-T.After Transformated in DH5αE. coli, white clonies can be picked for analysis. The Recombinant plasmid was named PT-F'and sent to Shenggong Company Limited for sequence analysis. Use the software of DNAETAR,DNAMAN to deal the data, and analyze the results. The results of Sequence analysis showed that the F gene of strain NA-1 were 1 672 bp long,encoding 553 amino acids. The 112~115 amino acid codon of F' gene is 112Gly-Arg-Gln-Gly-Arg-Leu117, which is fit the prediction experimental result.The F' protein gene of NA-1 strain of Goose paramyxovirus was expressed by the new Baculovirus expression system- Bac to Bac expression system. The recombinant plasmid pT-F' and transposition vector pFastBacⅠfrom experiment 1 were cutted by restriction enzyme EcoRⅠand HindⅢrespectively. F' gene and linear pFastBacⅠwere got by agarose gel electrophoresis, recovery and purification. The positive recombinant plasmid PFF' was got after the recovered linear pFastBacⅠ and F' were linked by cohesive end using T4 DNA ligase. After PFF' was transformed into e.coli DH10Bac, F' was transpositioned to recombinant Bacmid plasmid to be recombinant Bacmid plasmid (re-Bacmid) by the assistant of helper plasmid. The white colony was selected on the agar plate including X-gal/IPTG, gentamicin, kanamicin and tetrycycline. Sf-9 insect cell Sf-9 was transfectioned by re-Bacmid which is extracted from the amplified colony. Re-Bacmid was copied, expressed, assembled to be recombinant baculovirus in insect cells, and so F' protein was expressed. The molecular weight of F' protein was 63KDa after it was identified by SDS-PAGE, western-blot and indirect immunofluorescence, and it also shows that F' protein was partly glycosylationed in the insect cell, the proportion of F' protein was about 5% of all the proteins in the insect cell. It is obvious that the F' protein has efficient immunogenicity after it was mutationed.