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.

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.

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.

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.

[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.

[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.

[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.

[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.

[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.

[Establishment of indirect ELISA diagnose based on the VP1 structural protein of foot-and-mouth disease virus (FMDV) in pigs].

The complete gene encoding the structural protein of FMDV(VP1) was subcloned into expression vector pPROex-HT, resulting in the fusion expression plasmid pPROexHT-VP1. After transformed into E. coli BL21(DE3) and induced by IPTG, the fusion protein was expressed in high level.

[Development and characterization of monoclonal antibodies against VP1 protein of AsiaI type foot-and-mouth virus].

Aim: To prepare the monoclonal antibodies against VP1 protein of type AsiaI foot-and mouth disease virus (FMDV) and identify the characterization.

Methods: Three cell of hybridization that strains secreted monoclonal antibody(mAb) against type AsiaI FMDV were produced by fusing mouse myeloma cells (Sp2/0) with spleen cells from BALB/c immunized with the purified recombinant VP1 protein.

Results: The three hybridized cell lines reacted with cattle type AsiaI FMDV, the titres of ascetic fluids of the three mAbs ranged from 1:10(5) to 1:10(6) indirect ELISA showed.

[Genome sequencing and analysis of foot-and-mouth disease virus Asia1/YNBS/58 strain].

The full-length genomic sequence of foot-and-mouth disease virus (FMDV) Asia1/YNBS/58 strain was determined by RT-PCR and compared with other 17 reference strains. The results showed that the complete genome of Asia1/YNBS/58 was 8164nt long including a 1061-nt 5' untranslated region (UTR), a 6990-nt open reading frame (ORF), and a 113-nt 3'UTR. The homology analysis indicated that the UTR regions and non-structural proteins were more conserved than the structural proteins in FMDV.