Cloning, Expression Of Gp51 Gene Of Bovine Leukemia Virus (BLV) And Preliminary Development Of ELISA For Antibody Detection

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis (EBL),is characteristic by the increasing of lymphocytes number. EBL has been spread to almostall over the world and the prevalence of the infection rate in some cattle herds could be upto 60%. EBL is becoming one of the important cattle infectious diseases which produceenormous influences on the cow industry development. In this study, the structural proteingp51 gene of BLV was amplified from the BLV genome by PCR and the recombinantgp51 protein was expressed effectively in E. coli BL21 (DE3). Using the purified gp51protein as coating antigen, an indirect ELISA method was successfully developed fordetecting anti-BLV antibody in the bovine serum. The establishment of gp51-ELISAprovided us a rapid diagnostic method of BLV. The results of the research are as follows:1. The virus strain of FLK-BLV was grown in Minimum Essential Medium (Containing10% FCS). The gp51 gene was amplified by PCR using the viral genomic DNA astemplate. The specific product of gp51 gene was cloned into the pCR-Blunt Vector forsequencing. The sequence was analyzed by DNASTAR program. The results-showed thatgp51 gene consists of 804 bp fragment, which shared 100% homology with the sequencestrain that Noriyuki Sagata reported.2. The gp51 gene was cloned into pET-32a vector to construct recombinant expressionvector (pET-32a-gp51), and then the recombinant vector was transformed into the hostbacteria BL21 (DE3). The expression of gp51 was induced by IPTG and then identifiedby SDS-PAGE. The immunocompetence of gp51 protein was evaluated by Western-blot.The purified soluble BLV-gp51 protein was obtained by treatment with progressivedecreased urea solutions and dialysis;3. The purified BLV-gp51 protein was used to establish an indirect ELISA method forthe detection of antibodies against BLV. After selecting the conditions with the optimaldilution of antigen, the best coating concentration of gp51 protein was 2.19μg/mL, theoptimal dilution of primary antibody (serum sample) was 1:80 and the optimal dilution ofsecondary antibody (HRP-labeled rabbit anti-bovine serum) was to be 1:750. The analysisof indirect ELISA cross specificity revealed the negative reaction with positive sera ofinfection bovine rhinotracheitis(IBR), bovine viral diarrhoea(BVD), bluetongue(BT),akabane disease(AD), bovine serum uninfected with bovine leukemia virus(BLV) and apositive reaction with standard positive serum against enzootic bovine leukosis. It wasfound that the positive ratio was 11.63 % by detecting 172 bovine serum samples.Summary:①the gp51 gene of BLV was cloned successfully and it shared 100%homology with exist sequence of the reference;②the fusion proteins about 43 kD of gp51gene of BLV was expressed and it could react with the polyclonal antibody of bovine leukemia virus by Western-blot. The purified soluble BLV-gp51 protein was obtainedafter treatment;③an indirect-ELISA assay with high specificity, good sensitivity andeasy manipulation was established, which provided an available technique for detectionand serological survey of EBL.