| Orf is the anthropozoonosis caused by Orf virus belonging to the Parapoxvirus. Infection of Orf virus can interfere goats and sheeps with ingestion and suck to affect weight gain or lead to death by the secondary infection, thus causing serious economic loss to goat industry. It is widely acknowledged that humoral immunity plays an important role in Orf immune defense. Therefore the establishment of Orf antibody ELISA detection method is very important for evaluating immune effect of Orf vaccine and investigating Orf epidemiology.Orf virus major envelope protein B2L, heparin binding protein F1L and interferon-resi stance protein VIR are selected as coating antigen candidates of indirect ELISA because of their highly conservative property in different orf virus strains and function of stimulating infected goats producing anti-orf virus antibody. The primers were designed in accordance with announced B2L, F1L and VTR gene sequences in NCBI, and then PCR products of those genes were connected to the pGEM-T easy vector and transformed into DH5a cell, and then, recombinant plasmids were sequenced. The neucleotide and amino acid sequence identity were determined by DNA star, signal peptide were analysed by SignalP version3.0, and rare codon were predicted by rare codon forecast website. The exactly sequenced genes were double digested and subcloned into pET-32a expression vector and transformed into the competent cells BL21(DE3) to express proteins, then the soluble recombinant proteins were purified by Nickel column. Anti-recombinant proteins polyclonal antibodies were obtained by immuning recombinant protein from intradermal or subcutaneous of both sides of the spine and groin of the rabbits. The antigenicities of obtained Orf virus B2L, F1L and VIR proteins were evaluted by Western blot and Agar double diffusion test, antibody titers were determined by indirect ELISA method. The expressed proteins and Orf virus were used as coating antigen to detect53goat serum from adult goats and newborn lambs already eating colostrum. By comparing positive rate of recombinant proteins and Orf virus, we determined the best coating antigen and established indirect ELISA method, which can monitor Orf epidemic situation and evaluate vaccine immunity effect. The results were as follows: 1.Orf virus conservative genes B2L, F1L and VIR were successfully cloned and the obtained fragments of B2L, F1L and VIR were1137bp,1011bp and552bp. The results of sequence analysis indicated that B2L, F1L and VIR genes respectively shared96.7%-98.0%,97.3%and94.9%-96.2%identity to gene sequences announced in NCBI, and amino acid sequence identity reached96.0%-97.9%,91.8%-92.9%,96.7%-97.3%, which illustrated that these three genes were highly conservative in different orf virus strains.2.The prokaryotic expression products of B2L and VTR genes were obtained. The highest expression of pET32a-B2L-BL21was induced for4h at10℃when concentration of IPTG is0.3mmol/L in supernatant, and the highest expressing amount of pET32a-VIR-BL21in supernatant was induced for11h at37℃when concentration IPTG is1mmol/L. The antibody titers of B2L and VIR proteins evaluated by indirect ELISA method were1:200000and1:100000, respectively. The result indicated that B2L and VIR proteins can specifically bind with rabbit-anti orf virus antiserum and the positive serum of orf virus by Western blot and Agar double diffusion test.3.The best reaction conditions of B2L protein, VIR proteins and orf virus as an indirect ELISA coating antigen were identified. The best coating concentrations of B2L protein, VIR proteins and orf virus respectively were22μg/mL,30μg/mL and106.2TCID50/mL. The best coating liquid is carbonate buffer solution, and the best time of first antibody for B2L protein, VIR proteins and orf virus respectively were40min,40min,60min.The best time of second antibody for B2L protein, VIR proteins and orf virus all were60min, and the best time of TMB for B2L protein, VIR proteins and orf virus all were20min. The positive rate of53goat serum from adult goats and newborn lambs having to eat colostrum detected by indirect ELISA method indicated that the detecting result of B2L protein was the same as orf virus, that is56.6%(30/53). Therefore, Orf antibody ELISA detection method were successfully established by B2L protein as coating antigen.4. B2L protein had no cross reaction with goat pox standard positive serum, goat foot-and-mouth standard positive serum by the specificity test, indicating that Orf antibody ELISA detection method had high specificity.Orf standard positive serum diluted to1:105is still positive by the sensitivity test, indicating that Orf antibody ELISA detection method established had strong sensitivity. The result of Orf antibody ELISA detection method for detection of a copy of standard positive and negative serum,4undetected serum by intra-group and inter-group repeatability demonstrated that variable coefficient of intra-group and inter-group repeatability was0.23%-4.32%and1.59%-5.83%, less than15%, indicating that the detection method was repeatable. The clinical test indicated that Orf antibody ELISA detection method could be used for surveying Orf epidemic situation and evaluating vaccine immunity effect.In summary, we successfully cloned Orf virus conservative genes B2L, F1L and VIR, obtained B2L and VIR soluble proteins and finally established the Orf antibody ELISA detection method by using B2L protein as coating antigen. This newly established method can be used for surveillance of Orf epidemic situation as well as evaluation of vaccine immunity effect. |
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