| Infectious laryngotracheitis(ILT) is an acute, highly contagious respiratory disease caused by infectious laryngotracheitis virus (ILTV), it has caused a huge economic loss to the poultry industry. Currently, the control of the disease is mainly relied on live-attenuated vaccines at home and abroad. However, the live-attenuated vaccine has following disadvantages, such as the virulence remained strong, causing latent infection and regain virulence after consecutive transmitted in chickens. Epidemiological studies of ILTV among parts of the country found that the outbreaks of the disease may be related to live-attenuated vaccine. Therefore, the development of safe and effective genetically engineered vaccine has become a consensus.In this study, ILTV gB and gD gene were amplified from ILT Anhui-2011 strains, cloning the gene into the pUp-Down transfer vector containing US2 homology genes of HVT FC-126, to constructed the transfer plasmid pUp-gB-Down, pUp-gD-Down. Then cloning the expression cassette containing LTR promoter and EGFP reporter gene into these two transfer plasmids, to constructed the HVT transfer vector pHVT-gB, pHVT-gD. The recombinant virus with green fluorescent was produced by calcium phosphate-mediate transfection of CEF cells with the HVT genomic DNA and transfer vector. Using Cre recombinase knockout report EGFP, once again transfected CEF cells with recombinant virus genome, obtain the final recombinant virus rHVT-gB, rHVT-gD with only LTR promoter and gene gB, gD. Using PCR and Western-Blot test for identification, the results showed that the target gene is inserted into the HVT genome, and the virus can express gB and gD protein.The immune protective efficacy test of recombinant virus against ILT:901-day-old SPF chickens were randomly divided into five groups, the first and second group were recombinant virus immune groups, the third group was the challenge group, the Fourth group was ILTV attenuated vaccine group, the fifth group is the blank control group. In addition to the blank control group, the experimental groups were intratracheal inoculation of 105 EID50 ILTV virulent three weeks after ILTV attenuated vaccine immunization, continue observed for 10 days. Statistical the morbidity, clinical symptoms, antibody dynamics and histopathological changes of each group. The results showed that:Clinical symptoms appeared at two days after challenge, and became a peak after four days. The morbidity and mortality of rHVT-gB group were 20% and 5%, was more effective than the attenuated vaccine group, and the difference was significant (p< 0.05). The morbidity and mortality of rHVT-gD group were 30% and 15%, was more effective than the attenuated vaccine group, but the difference was not significant (p> 0.05). The result indicated that 1-day-old chickens inoculated with the recombinant virus can be produced effective protection against ILT virulent strain, and rHVT-gB was more effective than rHVT-gD group in immune protection.The immune protective efficacy test of recombinant virus against MD:601-day-old SPF chickens were randomly divided into four groups, the first and second group were recombinant virus immune group, the third group was FC-126 vaccine group, the forth group was the challenge group. Each experimental group were inoculated intraperitoneally 5000 PFU MDV virulent strain RB1B one week after immunization, continue observed until 90-day-old after challenge. The results showed that the recombinant virus can produce effective immune protection against MDV virulent strain like the parental strain FC-126, indicating that the insertion of foreign genes doesn’t affect the immune protection of parental strain. |
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