Construction Of Replication-Defective Adenovirus Containing The P1-2A And 3CD Gene Of Asia-Ⅰ Type FMDV And IL-18 Gene

Food-and-Mouth disease (FMD) is one of the most important animal diseases, which can cause a highly contagious disease affecting cloven-hoofed animals. It is redeemed as the first animal disease by the world OIE and our country .For providing the best products we constructed a recombinant replication-defection human adenovirus serotype 5 vector containing FMDV capsid, P1-2A, and viral 3CD protease coding regions and IL-18 gene ,which can give some adivise for the research of FMD adenovius vector vaccine. Research as follow:1) We cloned successfully the P1-2A and 3CD gene and IL-18 gene of Asia-I type FMDV.By mensurating the sequence we found that P1-2A gene has 2247 bp , which codes 749 amino acids .3CD gene has 2052bp including TAA , which codes 683 amino acids . we found that they have a high sequence resembling with IND01 and yunan baoshan FMDV sequence .They belong to the same subtype.2) we successfully cloned objective genes into adenovirus pShuttle-CMV vector. On the base of the measured sequence we designed the special primer, which have the special enzyme location . Subsequently we cut the objective genes and adenovirus pShuttle-CMV vector and connected them and transformed them . we distilled the plasmids and identified them .when constructed, the shuttle vector is linearized with Pmel and contransformed into BJ5183 together with pADEasy-1, the supercoiled viral DNA plasmid. Transforments are selected for Kanamycin resistance, and recombinants are subsequently identified by restriction digestion. Once a recombinant is identified, it is produced in bulk using the recombination deficient DH5a strain .3) Purified recombinant Ad plasmid DNA is digested with PacI to expose its inverted terminal repests( ITR), and then used to transfect AD-293 cells where deleted viral assembly genes are complemented in vivo. The cell CPE could be observed within one week after transfected, and complete CPE appeared within 24-48h after four round passages. RT-PCR demonstrated that there were expression of the cloned genes. A PCR method has demonstrated that this recombinant could be stably passaged, and the 10th recombinant adenovirus concentration is 10^6.61TCID₅₀/0.lml.4) we carried the primary immunity efficacity test in Guinea pigs, we used the treated and stably passaged and high concentration recombinant adenovirus to immuned Guinea pigs . later exsanguinating termly and mensurating serum antibody . when the Guinea pigs produced high concentration serum antibody, we used the same tupe FMDV to attack ,by which we can find the protection efficacity of recombinant adenovirus.