| H9 subtype avian influenza virus (MV) is widely distzibuted and has resulted in considerable economic loss to poultry industiy in China in recent years. In order to protect poultry against this low pathogenic subtype of API, inactivated vaccines in oil emulsion have been developed and proved to be effective in reducing severity of disease and spread of MV in field situation. However, the use of inactivated vaccines was restricted because of the disadvantages they inherited, including higher cost and interference of routine surveillance by serological test Here, we describe the construction of recombinant fowlpox viruses, rFP V-HA and rFPV-HA-IFN- II, expressing hemagglutinin (HA) from subtype H9N2 of API and coexpressmg the HA and chicken type II interferon(IFN- II) respectively, and their protective efficacies against homologous challenge in chickens for the development of better vaccines.1.Cloning and analyzing the HA cDNAThe genomic RNA of H9N2 subtype AIV AlChickenlCbina/F11998 (F strain for abbreviation), was extracted by phenol-SDS. When the purified genomic RNA was used as template for cDNA s~thesis and RT-PCR with two pairs of primers, two overlapping partial segments covering the complete HA cDNA, having the length of 1.1Kb and 0.8Kb nucleotides respectively, were obtained. Sequencs of the two eDNA segments showed that there was a unique Pst I site in the overlapping region of these two segments. By use of the unique Pst I site, we ligated these two segments into the complete HA cDNA, which was cloned in the recombinant plasmid pUCHA.vi~H )~f#眫{~The cloned HA gene was 1708 nucleotides long with a single open reading frame of 1680 nucleotides by sequence analysis. The open reading frame encoded a polypeptide of 560 amino acids, which included the signal peptide of the initial 18 amino acids at the N-terminal and the remaining segment of 542 amino acids. The latter was cleaved into two peptides, HAl and HA2, at the 320 amino acid site. The amino acids ofF strain at the cleavage site were RSSR ~ CILF, revealing the typical cleavage site features of low pathogenic AIV Compared with the H9 prototype AJV strain, A]Turkey/Wisconsin/1/66, the HA gene ofF strain shared 82.90/a sequence homology with that of it.According to the phylogenetic tree established for the H9N2 AJV isolates in China and neighboring countries and regions from 1992 to 1999, these isolates formed three lineages. F strain, the isolate from Beijing in 1994 and some 9 isolates from Hong Kong in recent years formed the first phylogenetic lineage, which showed significant phylogenetic difference with another lineage consisting of Korea isolates. The third lineage, also formed by isolates from Hong Kong, bad close phylogenetic relationship with the first lineage.2.Construction of the rFPV expressing HA (rFP V-HA)For the construction of rFPV-HA, the HA gene, removed from pUCI-IA as Sal I-Hind III fragment, was directionally inserted into the plasmid 1175, resulting in the transferring vector 11 75HA, in which the HA gene was under the transcriptional control of the vaccinia promoter P7.5. Then the transferring vector 11 75HA was used to transfect the chicken embryo fibroblast cells (CEF) pre-infected with wild type FPV (wt-FPV). The recombinant viruses were obtained and purified by blue plaque selection. The presence of HA gene in the genome of the purified recombinant virus was assayed by PCR and the expression of HA in the rFPV-HA-infected CEF was detected by indirect immunofluorescence.3.Cloning and expressing the chicken IFN- II geneA pair of primers were designed on the published data to amplify chicken WN- II gene with the Con A-stimulated chicken spleen cells. The cloned WN- II gene was then directionally inserted into the Sal I-Hind LII site of the plasmid pAFO9, resulting in the recombinant plasmid AWN, in which the IFN- II gene was under the control of theH9 *5~~ HA ~ I N-ll~ Iffl~1~ viivaccinia promoter PE/L.For the amplification... |
PDF Full Text Request