Basic Study On The Recombinant Vaccine Of Infectious Bovine Rhinotracheitis(IBR)

Infectious bovine rhinotracheitis(IBR), bovine herpesvirus-1(BHV-1) infection of cattle, has a wide variety of clinical manifestations, known mainly as a kind of respiratory tract disease characterized by rhinotracheitis, conjunctivitis, fever, abortion, infectious pustular vulvovaginitis. IBR was first described in 1955 as a new respiratory tract disease of feedlot cattle in western United States. IBRV was recognized as a number of herpesvirus by Huck in 1964. From 1972, the more severe respiratory tract form of IBR also became widespread in western European countries. It was found from the imported breeding cattle in China in 1970s, and then the virus was isolated and recognized. Now various herds in many provinces and cities are infected by IBRV, furthermore, IBR tends to rise gradually, and has profound effects on feed efficiency, milk production and reproduction.According to the published IRs repeat sequence of IBRV strain K22, a pair of primers was designed and synthesized.DNAs from five strains of different origins were amplifed by PCR, and a unique DNA fragment of about 540bp was obtained and cloned into pMD~18T vector. Both the nucleotide sequences and amino acid sequences were compared with those of K22 strain. It was showed that the homology of the nucleotide and the deduced amino acid sequence of the six strains were more than 98%.By means of PCR, the gene encoding gD of the strain Luojing of BHV-1 was amplified, sequenced, and cloned into plasmid pcDNAS. 1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100ug of the only plasmid or together with liposome. Serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibodies by ELISA. It was showed that the plasmid encoding IBRV gD developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.Vaccination is one of the most important measures in the prevention and control of IBR. In some countries, modified-live and killed IBRV vaccines have been used to control the disease. However, these conventional vaccines areusually remained insufficient attenuation and have shown a variety of sides effects including spread of vaccine virus to nonvaccinates, occurrence of long-term "carriers" . therefore, the long-term use is disadvantageous in attempts to eradicate IBR. With the improvement and use of recombintant DNA technology, people have been seeking more efficient geneic engineering vaccines, of which the live vector vaccine is one of the most active areas. In this report, bovine herpesirus-1 strain LA DNA Hindlll A was digested by the restriction endonuclease Sail. The Sail subfragment of 2. 7kb containing TK gene was cloned into pBluescript SK, resulting in a recombinant pTK. Then it was digested by Bglll and SacI, the 5.31kb fragment was recovered and self-ligated, resulting in a recombinant pdTK, the multiple coining sites were removed by Hindlll and Xbal; A fragment containing the immediate early promoter of cytomegalovirus, multiple cloning sites and bovine growth hormone polyadenylation signal derived from pCR3-IM was amplified and inserted into the Xhol site of pdTK. The resulting transfer vector pdTK-CMB can be used to expess genes from other bovine viruses (e.g. BPIV3> BRSV, BVDV) .Bovine ephemeral fever (BEF) is an acute febrile infection of cattle and water buffaloes and is caused by an arthropod-borne rhabdovirus. The infection occurs in many tropical and the America, and is recognized to be of major enconomic importantce in Australia, China, Japan and South Africa. The transmembrane glycoprotein G is the major neutralizing and protective antigen of BEFV. The BEFV JB76H G gene and LacZ gene were inserted individually into the universal IBRV pdTK-CMB transfer vector, and the transfer plasmid pdTK~LacZ-G was thus constructed. It was used to transfect MDBK cells infected with IBRV using lipofectin. The recombintant virus was plaque-purified in the presence of X~gal for five rounds, and was identified by...