Study On Recombinant Newcastle Disease Virus Expressing S Gene Of Infectious Bronchitis Virus

To investigate the influence of rescue of virus expressing long fragment, and the preparation of recombinant Newcatle disease virus(NDV) containing infectious bronchitis viru(IBV), the gene encoding the spike (S) protein of IBV Massachusetts 41 strain was amplified by RT-PCR with specific primers and cloned into NDV rLaSota vaccine strain. The recombinant fowl poxvirus expressing T7 polymerase pre-infected cell was transfected with this plasmid by calcium phosphate transfection method with helper plasmids. The recombinant virus rL-IBVS was successfully rescued. By means of the above method, rL-IBVS1 was rescued by taking S gene as a template. On this base, we constructed another recombinant virus rL-FmF-S1 that expressed S1.Genetic stability, the expression of foreign proteins, biological characteristics and immunity test on SPF chickens were studied. Foreign gene in recombinant virus was detected by RT-PCR. BSR cells infected with recombinant virus also showed high sensitivity in detection of specific serum by indirect immunofluoresence (IFA), confirming that the foreign protein was expressed. The results of MDT, ICPI and IVPI demonstrated rL-IBVS and rL-IBVS1 replicated to a titer similar to that of parental NDV LaSota strain in chicken embryoes and kept the low pathogenicity similar with LaSota vaccine strain. The ICPI value of recombinant virus rL-FmF-S1 was 0.52, slightly higher than that of rL-IBVS and rL-IBVS1. The IVPI value of rL-FmF-S1 still keeps 0.00. The value of MDT for rL-FmF-S1 was 110.3 h, greater than 90 h. Introduction of a foreign gene into NDV genome resulted in growth retardation and attenuation. Immunity test results showed that three recombinant viruses were effective in stimulating the body to generate antibodies. The recombinant virus constructed by applying reverse genetic techniques provided the basis for the development of a novel live viral vector vaccine to prevent both IB and ND.