Immunogenicity Of M And N Gene Of Avian Infectiousbronchitis Virus H120

Avian infectious bronchitis(IB) is an acute and highly contagious disease caused by theinfectious bronchitis virus(IBV) that can infect chickens at all ages. It is one of the most economically important diseases in poultry industry and used to be ranked in list B oftransmissible diseases by the World Organization for Animal Health. New types of IBV continue to arise due to mutations and recombination events in the viral genome, making this virus difficult to identify and extremely difficult to control. This study constructed eukaryotic expression plasmid based on the nucleocapsid(N) and the membrane(M) protein genes of IBV H120 and also tested their immune ability. The results could provide more information on the research of DNA vaccine against IB.1.IBV genome encodes three major structural proteins, of which the N protein(IBV-N) is well-conserved across various Ibv strains and thought to participate in RNA synthesis; the M protein(IBV-M),thought to be meaningful in virus replication, interacts with the nucleocapsid in the formation of closed viral particles and determines the sites at which virus particles are assembled. IBV-M is less conservative than IBV-N but both have importantantigenic determinants. In this study, the gene sequence of IBV H120(GenBank accession no. FJ888351.1) was retrieved from GenBank.The protective antigen gene of I BV-M and IBV-N were amplified by RT-PCR technology with a pair of primers designed usingPrimerPremier5.0 according to the M gene and the N gene of IBV. DNA sequencingindicated that the M gene had about 678 bases,resulting in 225 amino acids;while the N gene had approximately 1230 bases,resulting in 410 amino acids.Compared with other regisitered strains in GenBank,they presented little mutation.the homogeneous rate for M gene was between 84% and 100%;with the corresponding homogeneity of amino acid between 86% and 100%;for N gene the homogeneous rate was between 90.2% and 100% and the corresponding homogeneity of amino acid was between 91% and 100%.2. The eukaryotic expression vector of p EGFP-C1 was modified by SCP strong promoterand SEC sequence to form a new vector, namely p EGFP-C1-SCP-SEC, which was then combined with the amplified genes of IBV-M and IBV-N after their double digests with therestriction enzyme of Eco RI and SalI. The products were then transformed into thecompetent E.coli DH5α cells. After being screened and sequenced with PC R, the true positive clones were identified and the construction of the eukaryotic expression vector was completed. This recombined plasmid was transfected into Vero cells. The intracellular expression of M gene and N gene and their immunogenicity was detected byimmunofluorescence test, which built a promising foundation for further studies of DNA vaccine.3. The constructed eukaryotic expression vector was enclosed with liposome PEI25 000(N/P=8) to formulate a DNA vaccine, which was testedwith immunity tests by multi-point injections of the vaccine into the leg muscles of chicks. The first immunization was performed at 10 daysold and the reinforced immunization was at 21 days old. Then all vaccinated chicks were challenged with IBV H120. After 15 days of observation the virus was tested in the kidney tissues. The virus detection rate was 33.5% in the attenuated IBV- vaccinated group, 50% in the M plasmid- vaccinated group and 40% in the N plasmid-vaccinated group. This indicated that the singlegene- vaccinated groups had higher than the attenuated IBV- vaccinated group. However, the virus detection rate of the M and N plasmids-based group was as low as 20%, lower than both the attenuated IBV group and the single-gene group. These results showed that the PEI25000-enclosed DNA vaccine had an efficient protection against IBV and hence is meaningful for the control and prevention of IBV infection.