Study On DNA Vaccine Of M Gene Of Avain Infectious Bronchitis Virus (IBV_WHNJ Strain)

According to the M gene sequence of IBV on GenBank, a pair of specific sequences as primers designed to amplify the M gene of IBVwHNj isolation from Sichuan province in China. By reverse transcription polymerase chain reactin (RT-PCR),M gene of IBV was amplified successfully. The PCR products were cloned into PGEM-T vector and sequenced. Sequence analysis indicated that the length of gene coding for M of IBVwHNj is 678bp, and the deduced amino acid sequence length is 225aa. The nucleotide sequences and deduced amino acid sequences of the M gene of IBVwHNj were analyzed and compared with the published sequences of reference strains in GenBank, the results showed that the nucleotide sequences homogeneity is 91.6%~100%, and the deduced amino acid sequences homogeneity is 91.6%~100%. The nucleotide sequences of the M gene of IBVwHNj were submitted to Genbank, the accession number is AY3 02742.The M gene of IBVwHNj was subcloned into the eukaryotic expression plasmids pcDNA3.1 and the obtained recombinant plasmids were designated as pc-M. The cationic liposome were made and the recombinant plasmids pc-M were entrapped with cationic liposome by 10:1 and were observed by electron microscope. The results showed that the size of cationic liposome was 30-200nm and the size of compound was 50~400nm, the shape was anomalous. In this reseach, the DNA vaccine of pc-M recombinant plasmids containing M gene of IBVwHNj have been made and were entrapped with cationic liposome.In order to detect immune efficiency of pc-M, 10-day-old chickens were immunized by four sites intramuscular injection with the entrapment of plasmids with cationic liposome or pc-M, the dose of each injected plasmid for a chicken is 200 ug. The chickens were immunized with sameness dose after a week. The Veinal blood of all chickens were phlebotomized befroe immunized, the 24th day and the 31st day respectively. The dynamic changes of white blood cells and the antibody were detect and analyzed by SPSS. The results showed that recombinant plasmid DNA of M gene induced strong responses of humoral immunity and cell-mediated immunity. The number of white blood cells and the IgG content were significantly raised in the vaccinated groups while the control groups showed nochanges, the entrapment of plasmids with cationic liposome considerably elevated the cellular and humoral immune responses of vaccinated chickens in comparison with other groups. Challenges with 102EIDso dose of IBVwHNJ were administered at two weeks after the second DNA inoculation,the protective immunity was observed. After challenge, chickens in test group have no clinical symptom, three died in control group.The results indicated that the chickens immuned by DNA vaccine of M gene of IBVWHNJ could be against IBV effectively.