| Pseudorabies, c aused b y p seudorabies v irus (PRV), i s a d isease c haracterized b y neurological disorder in piglets and reproductive failure in pregnant pigs. The vaccines currently used in China and other countries are all with gE-phenotype. Swine influenza (SI) is a contagious respiratory disease or syndrome, characterized by severe respiratory disease in young pigs with almost 100% morbidity, abortions in breeding herds and a few deaths of adult pigs. Swine is the only animal which can be infected by either avian or human original influenza viruses, and c an be considered as the intermediate host for the process of genetic reassortments between viruses of different hosts. Therefore, vaccination against swine influenza has significant meaning for food safety and public health. The purpose of this study is to construct a recombinant pseudorabies virus expressing HA gene of swine influenza virus and to develop specific diagnosis for differentiating the recombinant virus immunized animals and those naturally infected with PRV or SFV.In the study, a sequence coding for HA of SFV subtype H3N2 (A/Swine/Inner mogolian/547/2001) was amplified by RT-PCR and sequenced. The gene is 1701bp in length encoding 566 amino acid residues. Sequence comparison indicated that the nucleotide homologies between the HA gene and those from eight other H3 subtype SFV isolates from China are over 95%, and nucleotide identities between H3 SFV isolates in China and a typical H3 subtype avian influenza virus(AIV) strain DKUK1-63 are 95% to 100%. These might tell us that the HA gene of all H3 subtype SIVes isolated in China was originally from AIV Although the vaccine based on HA do not protect against different subtype of influenza virus, it can provide favorable resistance to infection with antigenic variants within a subtype.To construct a transfer vector, PRV genomic DNA was extracted and digested by KpnI, and the KpnI fragment J containing the thymidine kinase (TK) gene was cloned into pUC119. hi further, a 2.6kb PstI-KpnI fragment with TK gene within Kpnl J fragment was subcloned and generated a recombinant pBTK2.6. The EcoRI site and Noti-HindIII fragment were deleted from the recombinant vector. And then a 378bp AccI fragment in pBTK2.6 was replaced by a fragment containing the immediate early promoter of cytomegalovirus and bovine growth hormone polyadenylation signal derived from pCR3-Uni, a eukaryotic expression plasmid. The recombinant vector was digested with Tthllll and the LacZ gene from E. coli was inseted in this site, the generated plasmid is designated as pLTK-Uni. Finally, a tranfer vector named as pLTK-HA was constructed based on pLTK-Uni with insertion of HA gene of H3 subtypesrv.The pLTK-HA and PRV Bartha-k61 genomic DNA were co-transfected into 50-70% confluent Vero cells in 6-cm dishes. Based on the expression of LacZ gene,recombinant PRV were selected and purified by blue-colored virus plaque. The recombinant PRV was identified by PCR and designated as rPRV-HA. The expression of HA in Vero cells infected with rPRV-HA was detected by Western-blot. The results indicated that HA protein could be consistently detected from a serial passages of Vero cells infected with rPRV-HA. The recombinant virus can be further developed as a live vectored vaccine against pseudorabies and swine influenza.To differentiate the animals immunized with rPRV-HA and those naturally infected with PRV or SFV in future, two differentiating diagnostic methods were developed based on recombinant viral antigens.Although dramatic variation occurs frequently in genome of type A influenza virus, the NP gene is comparatively conservative representing virus type specificity. In this study, a cDNA encoding for NP protein of a SIV isolate, A/Heilongjiang 73/2000 (H3N2), was amplified by RT-PCR, and cloned into prokaryotic expression plasmid pET30(a). The recombinant plasmid carrying NP gene was designated as pET-NP. After transformation of BL21 with pET-NP, an expressed fusion protein with molecular weight of 60Ku was identi...
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