Construction And Rescue Of Recombinant Newcastle Disease Virus Expressing Glycoprotein GB Of Infectious Laryngotracheitis Virus

Infectious laryngotracheitis (ILT) is an economically important viral respiratory tract infection of chickens, caused by infectious laryngotracheitis virus. The disease occurs worldwide and can result in severe production losses, characterized by signs of high mortality and decreased egg production. ILT was found in our country in the 1960’s and occurred in many provinces and cities one after another, causing huge economic losses to our country. Present vaccines for ILT are all modified live viruses. These vaccines remain insufficient attenuation and have shown avariety of side effects including spread of vaccine virus to nonvaccinates, occurrence of long term "carrier" birds, and increasing virulence during in vivo passage, and we can’t differentiate the vaccine strain from the field strain. Therefore, it is necessary to research new vaccine for the prevention of ILT. With the development of molecular biology, genetic vaccine is the best choice for preventing and eliminating ILT.Newcastle disease (ND) is a major deadly infectious disease endangering the poultry industry around the world. Because of the recombinant NDV’s genetic stability, a variety of immunization forms, low cost of production, safty and efficiency, we choose it as the carrier of the recombinant vaccine. Inducing both humoral and cellular immunity, protein gB is not only related to the infection and replication of the virus, but also necessary for ILTV’s adsorption and penetration. As a major protective antigen of ILTV, protein gB is the best choice for the construction of genetic vaccine.In this study, we choose freeze-dried vaccine of infectious laryngotracheitis Henan strains as template, and amplify ILTV gB gene by PCR, using a pair of primers which are designed on the basis of ILTV’s gene sequences. Then we consruct a recombinant plasmid expressing glycoprotein gB of infectious laryngotracheitis Henan strains, with the reverse genetics systems of NDV LaSota attenuated strain. We co-transfect the recombinant plasmid and helper plasmid pBS-N, pBS-P, pBS-L into BHK-21 cells, with the method of calcium phosphate transfection. Meanwhile, we inoculat 9-11 days’ old SPF chick embryo cysts with cell culture, harvesting allantoic fluid whose hemagglutinin (HA) test is positive after five days’cultivation. By the identification of RT-PCR, we confirm the successful rescue of the recombinant virus rLa-ILTVgB. Then we passage the recombinant virus by inoculating the chick embryo cysts, and confirm the protein gB is correctly expressed in the recombinant virus by laser scanning confocal. According to the international standards, the MDT of the recombinant virus is above 168 h, and the ICPI and IVPI of the recombinant virus both are 0, which reveals that the recombinant virus maintains low pathogenicity as the parent strain. In this study, the recombinant virus rLa-ILTVgB as the live vector vaccine of infectious laryngotracheitis shows grate protential, and all these results provide good foundation for further research.