تولید آنتی‌سرم چندهمسانه‌ای اختصاصی علیه پروتئین پوششی نوترکیب ویروس پنهان زعفران (SaLV)

Lack of availability to specific antibodies against Potyvirus croci (saffron latent virus (SaLV)) has hindered the development of the serological assay methods of SaLV. In this study, following SaLV coat protein (CP) gene cloning in pET28a (+) expression vector, the recombinant coat protein was expressed in bacterial system and purified through affinity chromatography using Ni-NTA resin. In order to production of the SaLV specific polyclonal antiserum, purified recombinant SaLV-CP was injected subcutaneously at two-week intervals to a female New Zealand White rabbit. Each injection contained one milliliter of the purified recombinant SaLV-CP (approximately 70-100 μg/mL) with an equal volume of Montanide adjuvant. After the third injection, a blood sample was collected from the marginal ear vein and kept at 37°C for one hour and centrifuged to separate the blood serum. In the Agar double diffusion test, the antiserum obtained displayed a clear band with purified recombinant SaLV-CP and SaLV-infected saffron plants extracts, while no band was observed with the extract from healthy plants. The antiserum was used in the indirect ACP-ELISA assay, resulted in a specific high reaction with different dilutions of purified recombinant SaLV-CP and SaLV-infected saffron plants extracts, with OD405 values ranged 5-10 times greater than that of the average absorption observed in healthy plants. Based on these results, the purified recombinant coat protein of SaLV had strong immunogenicity in the rabbit. The primary generated antiserum (followed by the third injection), exhibited specific and highly sensitive reactions to the virus in the earlier-mentioned serological tests. It is expected that the finally generated antiserum (followed by the fifth injection) and the purified antibodies exhibit reactions with high specificity, reproducibility, and sensitivity in the specific detection of SaLV. The raised SaLV-specific antibodies will be utilized to develop the rapid and specific serological detection systems of SaLV in the saffron fields, appropriate SaLV management strategies and determine the natural host range of the virus.