Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

BACKGROUND: In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. RESULTS: In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 µL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. CONCLUSION: We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

Develope monoclonal antibody against foot-and-mouth disease virus A type.

In order to develop an anti-FMDV A Type monoclonal antibody (mAb), BABL/c mice were immunized with FMDV A type. Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88. The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512, respectively. Both mAbs contain kappa light chains, the mAbs were IgG1. In order to define the mAbs binding epitopes, the reactivity of these mAbs against A Type FMDV, were examined using indirect ELISA, the result showed that both mAbs reacted with A Type FMDV. These mAbs may be used for further vaccine studies, diagnostic methods, prophylaxis, etiological and immunological research on FMDV. Characterization of these ncindicated that prepared anti-FMDV A mAbs had no cross-reactivity with Swine Vesicular Disease (SVD) or FMDV O, Asia1 and C Type antigens. Their titers in abdomen liquor were 1:5×10(6) and 1:2×10(6), respectively. 7B11 was found to be of subtype IgG(1), 8H4 was classified as IgG(2b) subtype. The mAbs prepared in this study, are specific for detection of FMDV serotype A, and is potentially useful for pen-side diagnosis.

Promising multiple-epitope recombinant vaccine against foot-and-mouth disease virus type O in swine.

In order to develop a completely safe immunogen to replace the traditional inactivated vaccine, a tandem-repeat multiple-epitope recombinant vaccine against foot-and-mouth disease (FMD) virus (FMDV) type O was developed. It contained three copies each of residues 141 to 160 and 200 to 213 of VP1 of the O/China/99 strain of FMDV coupled with a swine immunoglobulin G heavy-chain constant region (scIgG). The data showed that the multiple-epitope recombinant vaccine elicited high titers of anti-FMDV specific antibodies in swine at 30 days postvaccination (dpv) and conferred complete protection against a challenge with 10³ 50% swine infective doses of the O/China/99 strain. The anti-FMDV specific antibody titers were not significantly different between the multiple-epitope recombinant vaccine and the traditional vaccine (t test, P > 0.05). The number of 50% pig protective doses was 6.47, which is higher than the number recommended by the World Organization for Animal Health. The multiple-epitope recombinant vaccine resulted in a duration of immunity of at least 6 months. We speculate that the multiple-epitope recombinant vaccine is a promising vaccine that may replace the traditional inactivated vaccine for the prevention and control of FMD in swine in the future.

[Prokaryotic expression, protein purification and reactivity analysis of the VP1 and its C terminus of a South African isolate of FMDV serotype SAT2].

AIM: To induce the expression of structure protein VP1 and its C terminus of foot-and-mouth disease virus (FMDV) serotype SAT2 in E.coli and analyze their reactivities with FMDV positive antiserum. METHODS: The plasmid pGEM-SAT2P1 carrying the VP1 coding region of FMDV serotype SAT2 isolated from South African was used as template for RT-PCR to get the coding fragment of VP1 and its C terminus. The fragments were then cloned into expression vector pET-30a(+) to get recombinant plasmids pET-SAT2VP1 and pET-SAT2VP1C. The recombinant plasmids were transformed into E.coli BL21(DE3)pLysS and induced by IPTG to express VP1 and its C terminus protein. The expressed VP1 and its C terminus were then purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blot. New Zealand rabbits were immunized to prepare polyclonal antibodies against VP1 and VP1C. The antisera were obtained and polyclonal antibody was characterized by ELISA. RESULTS: SDS-PAGE demonstrated that VP1and its C terminus expressed in the E.coli transformants had a molecular weight of 33000 and 19000 and contained in the inclusion body. Purified VP1 and its C terminus was obtained by Ni-NTA His Bind Resin affinity chromatography and a single clear band appeared in the SDS-PAGE gel. Western blot analysis showed that the purified VP1 and VP1 C terminus could react with bovine antiserum against the same serotype FMDV without cross-reactivity with the negative bovine serum. CONCLUSION: Rabbit polyclonal antibodies against VP1 and VP1C were successfully prepared, the titers of which were above 1:12800 and had obvious specificity.

B cell epitopes within VP1 of type O foot-and-mouth disease virus for detection of viral antibodies.

In this study, the coding region of type O FMDV capsid protein VP1 and a series of codon optimized DNA sequences coding for VP1 amino acid residues 141-160 (epitope1), tandem repeat 200-213 (epitope2 (+2)) and the combination of two epitopes (epitope1-2) was genetically cloned into the prokaryotic expression vector pP(RO)ExHTb and pGEX4T-1, respectively. VP1 and the fused epitopes GST-E1, GST-E2 (+2) and GST-E1-2 were successfully solubly expressed in the cytoplasm of Escherichia coli and Western blot analysis demonstrated they retained antigenicity. Indirect VP1-ELISA and epitope ELISAs were subsequently developed to screen a panel of 80 field pig sera using LPB-ELISA as a standard test. For VP1-ELISA and all the epitope ELISAs, there were clear distinctions between the FMDV-positive and the FMDV-negative samples. Cross-reactions with pig sera positive to the viruses of swine vesicular disease virus that produce clinically indistinguishable syndromes in pigs or guinea pig antisera to FMDV strains of type A, C and Asia1 did not occur. The relative sensitivity and specificity for the GST-E1 ELISA, GST-E2 (+2), GST-E1-2 ELISA and VP1-ELISA in comparison with LPB-ELISA were 93.3% and 85.0%, 95.0% and 90%, 100% and 81.8%, 96.6% and 80.9% respectively. This study shows the potential use of the aforementioned epitopes as alternatives to the complex antigens used in current detection for antibody to FMDV structural proteins.

[Gene cloning of ligand binding domain of porcine beta3 as FMDV receptor and preparation of its polyclonal antibody].

AIM: To clone and express the ligand binding domain (LBD) cDNA of porcine integrin beta3 as foot-and-mouth disease virus (FMDV) receptor and prepare its polyclonal antibody. METHODS: The LBD cDNA of porcine beta3 was obtained from the lung tissue of pig infected with FMDV by RT-PCR, and the recombinant plasmid pGEM/beta3LBD was constructed. After digested with BamH I/Xho I, the beta3LBD fragment was subcloned into prokaryotic expression vector pGEX 4T-1. The recombinant expression plasmid pGEX/beta3LBD was constructed and transformed into E.coli BL21(DE3). The recombinant porcine beta3LBD protein was expressed after IPTG induction and purified from total protein of BL21(DE3). The rabbits from New Zealand were immunized with the purified fusion protein to prepare polyclonal antibody, which was identified by Western blot and ELISA. RESULTS: The 507 bp cDNA of porcine beta3LBD encoded a polypeptide of 169 amino acids. The similarity of nucleotide sequence beta3LBD between pigs and cattle, human being, chimpanzees, rhesus monkeys, horses, dogs, Norway rats, mice, chickens was 90.3%, 92.3%, 92.1%, 91.3%, 90.5%, 90.3%, 87.8%, 85.2%, 79.5%, respectively. The beta3LBD gene of mammals exhibited high sequence homology. The recombinant beta3LBD protein was expressed efficiently as inclusion body after IPTG induction and was approximately 44000. The titer of the polyclonal antibody against the purified beta3LBD protein was about 1:12 800 by ELISA. CONCLUSION: The gene cloning and expression of beta3LBD and the preparation of its polyclonal antibody lay a foundation for further research into the interaction of FMDV with beta3 subunit of porcine integrin.

[Preparation and characterization of the polyclonal antibody against pig integrin beta6 subunit LBD as FMDV receptor].

AIM: To induce the expression of FMDV receptor integrin beta6 subunit ligand-binding domain in E.coli and prepare the rabbit polyclonal antibody against it. METHODS: The fragment coding beta6 ligand-binding domain was amplified by PCR and doubly digested with BamH Iand Xho I. Then it was cloned into expression vector pGEX-4T-1 to obtain recombinant plasmid pGEX-4T-1-beta6LBD. After pGEX-4T-1-beta6LBD was transformed into E.coli BL21(DE3) and induced by IPTG, the expression of fusion proteins was identified by SDS-PAGE, with inclusion body prepared and fusion protein purified. Then new Zealand rabbits were immunized to prepare polyclonal antibody against beta6LBD. GST-beta6LBD antiserum was obtained and the specificity of polyclonal antibody was detected by Western blot. RESULTS: SDS-PAGE demonstrated that the fusion protein GST-beta6LBD was expressed with the expected molecular weight at 42 000. A single clear band of GST-beta6LBD fusion protein appeared in SDS-PAGE gel after purification. The titer of the polyclonal antibody was above 1:12 800 and it is of high specificity. CONCLUSION: The successful preparation of rabbit anti pig beta6LBD polyclonal antibody with high affinity and specificity will lay a foundation for further research into the function of integrin beta6 in FMDV infection.

[Cloning and sequence analysis of cDNA encoding porcine alphav subunit for FMDV receptor].

Receptors play a crucial role in determining the pathogenesis and tissue tropism of virus. Foot-and-mouth disease virus (FMDV) has been showed to use four integrins, alphavbeta1, alphavbeta3, alphavbeta6 and alphavbeta8 as receptors to initiate infection. In this study, the porcine integrin alphav gene was cloned by RT-PCR from the lung tissue of healed pig infected experimently with FMDV, and compared its nucleotide and deduced amino acid sequence with the av gene of other animals. The 3141bp cDNA of bovine integrin alphav encodes a polypeptide of 1046 amino acids consisting of a 30-residue putative signal peptide, a 955-residue ectodomain, a 29-residue transmembrane domain, and a 32-residue cytoplasmic domain. The ectodomain contains 11 potential N-linked glycosylation sites (NXT/NXS), 2 calcium binding domains (DX[D/N] XDGXXD) and 18 cysteine residues. The nucleotide sequence similarities of integrin alphav between pig and cattle, human, rheses monkey, house mouse, chicken, dog are 93.3%, 91.5%, 91.4%, 85.6%, 73.2% and 89.9% respectively; and the amino acid sequence similarities are 96.3%, 94.6%, 94.1%, 90.8%, 81.6% and 93.8%, respectively. The alphav gene of cattle and pig exhibited the highest sequence homology. It is possible that host tropism of FMDV may related to divergence in receptors among different species.

[Molecular cloning and characteristics of cDNA encoding pig beta6 subunit for FMDV receptor].

In order to study the roles of integrin beta6 in Foot-and-Mouth Disease Virus infection, pig integrin beta6 was firstly molecularly cloned from RNA of the tongue and lung of recovered pig infected experimentally with foot-and-mouth-disease virus (FMDV), and was compared with the beta6 gene of other animals available in GenBank at nucleotide and amino acid leves. GeneBank association number of the beta6 gene is EF432729. Pig integrin beta6 gene (2367bp) encodes a polypeptide of 788 amino acids consisting of 9 potential N-linked glycosylation sites, 3 Glycosaminoglycan attachment sites, a cGMP-dependent protein kinase phosphorylation site, 10 Protein kinase C phosphorylation sites, 2 EGF-like domains and 2 cysteine-rich regions. Pig integrin beta6 subunit has a 26-residue putative signal peptide, a 681-residue ectodomain, a 29-residue transmembrane domain, and a 52-residue cytoplasmic domain. 11 mutant nucleotides were found in beta6 gene coding region and 9 amino acids were changed. The nucleotide sequence similarity of integrin beta6 gene between rheses monkey, mouse, Norway rat, dog, guinea pig, human, bovine, sheep is 79.5%, 84.9%, 85.4%, 85.2%, 88.7%, 90.1%, 91.9% and 91.9%, and the amino acid sequence similarity is 93.5%, 88.2%, 88.5%, 88.3%, 91.0%, 92.8%, 93.3% and 93.4% respectively. This study will lay a foundation for understanding the interactions of FMDV with receptors.