| It is significative for plant virus detection,quarantine and scientific prevention and control to produce specific antibodies against important plant viruses and develop effective serological methods.Citrus tatter leaf virus causes an important viral disease on citrus in China after Citrus triteza disease and exocortis.Banana bunchy top virus brings bunchy top disease.which is an important viral disease,and the disease threatens about a quarter of the banana production of the world in Asia,Africa and The South Pacific.In this study,monoclonal antibodies (MAbs) against CTLV and BBTV were respectively producted.and serological methods based on the MAbs were developed for rapid,reliable and sensitive virus detection.(1) MAbs against CTLV and serological method for CTLV detection:The coat protein (CP) gene of CTLV was amplified by PCR and inserted into a prokaryotic expression vector pET-28a to produce pET-28a-CP, which was transformed Escherichia coli BL21(DE3). After induced by IPTQ an about30kDa fusion protein was expressed and purified through a Ni+-NTA affinity column. The purified recombinant CP was used to immunize BALB/c mice for producing MAbs. Three hybridoma cell lines secreting MAbs against CTLV were obtained. The result of TAS-ELISA showed that tissues. A dot-ELISA method based on MAbs was developed for CTLV detection, and this method could successfully detect the virus in infected tissue sap diluted at1:640(w/v,g/mL).(2) MAbs against BBTV and serological method for BBTV detection:After selected and cloned, one hybridoma cell line secreting MAbs against BBTV was produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized by purified BBTV particles. Ascites containing MAb against BBTV were prepared with the hybridoma. The MAb22E3could strongly react with BBTV particles and BBTV-infected banana plant tissues, not with the healthy banana plant tissues.Based on the MAbs22E3, a dot-ELISA for BBTV detection was developed. The dot-ELISA could specifically detect the virus in infected banana plant tissue extracts diluted at1:320(w/v,g/mL). |
