Expression Of Foot-and-mouth Virus(FMDV) Empty Capsids Antigen And The Assessment Of Its Immunogenicity

Foot-and-mouth disease (FMD) is one of the most important known pathogens of livestock. In 2005, the disease caused by foot-and-mouth disease (FMDV) serotype Asia I broke out in part of China. But the available research data of type Asiaâ… is relative lack. In this research, we have sequenced the intact open reading frame (ORF) of FMDV Asiaâ… /JQ strain, and expressed the empty capsids antigen using silkworm- baculovirus expression system.1. The intact ORF sequence of Asiaâ… /JQ strain was determined. It is 6987nt long and can be divided into four parts of L, P1, P2 and P3. With the help of the DNAStar ware, these four parts of Asiaâ… /JQ strain were compared with 7 reference strains. The comparison of the P1 confirmed that Asiaâ… /JQ has a high identity with type Asiaâ… reference sequences. But the identities of nonstructural proteins coding region were higher with type O strains than type Asia I strains. The phylogentic tree obtained from the nucleotide sequence of P1 gene from Asiaâ… /JQ strain and 7 reference strains shows that the Asiaâ… /JQ strain has a close relationship with the type Asia I strains. The phylogentic trees obtained from the nucleotide sequence of nonstructural protein coding regions show that the Asiaâ… /JQ strain has a close relationship with the type O stains. The results suggest that the gene recombination had occurred between 0 type strain and Asiaâ… type strain to recombine the Asiaâ… /JQ strain.2. The secondary structure of capid protein of Asiaâ… /JQ strain was predicted by the methods of Garnier-Robson, Chou-Farplus and Karplus-Schulzbased, and hydrophilicity plot, surface probability plot and antigenic index were obtained by the methods of Kyte-doolittle, Emini and Jameson-Wolf respectively. Combining the resultes according to these methods, the B cell epitopes of capsid protein of FMDV were predicted. The results showed that there is much flexible region such as coil region and turn region in capsid protein of FMDV, and more B cell epitopes in VP1, VP2 and VP3 than VP4. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of FMDV.3. The intact ORF of foot-and-mouth disease virus (FMDV) Asiaâ… /JQ strain was inserted into the transfer vector pVL1393 to generate plasmid pVL-ORF. The recombinant silkworm baculovirus rBmNPV(ORF) containing the intact ORF of FMDV was obtained after co-transfection and screening. Indirect immunofluore- scence test was used to verify that rBamNPV(ORF) could validly express the target cassette in Bm-N cell. The early 5th instar larvae of silkworm were infected with the recombinant virus. The polyprotein of FMDV expressed in silkworm was confirmed by sandwich-ELISA and empty capsid like particles could be observed in haemolymph. Expression products from silkworm were used as antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. Challenged with 10,000 BID₅₀ of virulent homologous virus, two of the four were completely protected, and clinical symptoms were alleviated and delayed in the others.4. The recombinant silkworm baculovirus rBamNPV(P1-2A3C) which contained the intact P1-2A and 3C protease coding regions of Asiaâ… /JQ stain was obtained. Indirect immunofluorescence test was used to verify that rBmNPV(Pl-2A3C)could validly express the target cassette. The sandwich-ELISA confirmed that the quantity of FMDV antigen in haemolymph was about 300 microgramme or more. Expression products from silkworm were used as antigen to immunize the cattle. The specific antibody was induced in all vaccinated animals. Challenged with virulent homologous virus, four of the five were completely protected, and clinical symptoms were alleviated and delayed in the other one.5. We followed the bovine potency test protocol described by the OIE to test this empty capsids vaccine potency. The PD50 (50 % protective dose) potency were detected on cattle with 10,000 BID₅₀ of virulent homologous virus. The results showed the vaccine potency of the batch immunized with the expressed antigens diluted 40 folds could get 3.60 PD₅₀ per dose and the vaccine potency of the batch with the expressed antigens diluted 30 folds could get 6.34 PD₅₀ per dose. These experiments lead to a conclusion that it is feasible to make use of silkworm- baculovirus expression system for FMD vaccine production.