Establishment Of Gold Immune-chromatographic Assay For Detecting Bovine Leukemia Virus-Specific Antibodies

Bovine leukosis (BL) is a chronic lymphoproliferative disorder in cattle caused by bovine leukemia virus (BLV). BL has a worldwide distribution with infection rate of 50-60% in some cattle herds. BLV causes heavy economic losses to the cattle industry due to condemnation of infected cattle, reduction of milk production and reproductivity, and loss of competebility for trade and export. Presently, no effective vaccine against BL is available and the control of the disease depends exclusively on eradication program. Therefore, there is urgent need for developing quick diagnostic tools with high specificity and sensitivity.The GP51 is the major and conserved envelope protein of BLV, which is commonly used as the target antigen for diagnostic purpose. To establish a BLV-specific antibody detection methed, the gp51 gene was amplified from the DNA extracted from BLV-infected fetal lamb kidney (FLK) cells by PCR. After confirmation of its correctness by sequencing, the PCR product was inserted into prokaryotic expression vector pET-32a and the resulting vector pET32a-gp51 was transformed into BL21 (DE3) E.coli. After induction with IPTG, the expected His-GP51 fusion protein of 42kDa was detected in the cell lysate of the recombinant E.coli by SDS-PAGE. Western blotting showed that the expressed protein reacted positively with the anti-BLV serum. His-GP51-containing inclusion bodies were isolated from the recombinant E.coli and submitted to affinity purification with Ni columns following denaturation with 8M urea. SDS-PAGE showed that the His-tag protein was purified to a single band, which was usable for establishing serological methods for inspection of BLV infection.The purified His-GP51 protein and goat-anti-bovine IgG were spotted onto nitrocellulose filter as the test line and the control line, respectively. Goat antiserum specific for the Fc fragment of bovine IgG (GAB IgGFc) was labeled with the optimized amount (6μg/ml) of colloidal gold and indirect colloid gold immuno-chromatographic assay (GICA) was established for detection of BLV-specific antibody. Using the colloidal gold dipstick, BLV-positive serum could be disgnozed in 10 minutes. Nine BLV-positive and 13 BLV-negative bovine sera were tested in parallel with the colloidal gold dipstick and commercial ELISA kit with a coincidence of 88.89%(8/9)for the positive sera or 84.62%(11/13)for the negative sera.