| T-2 toxin is one of the most toxic trichothecenes toxins produced by various species of Fusarium. Widely distributed in nature, T-2 toxin is dangerous food contaminants and risk to human and livestock health in various countries. Thus, phage library against T-2 toxin was constructed and higher affinity of scFv was screened which lay the basis for the detection of T-2 toxin. In addition, the cytotoxic effect of T-2 toxin was studied.In order to construct a single chain fragment variable (scFv) phage display library against T-2 toxin, which will laid the foundation for the detection of T-2 toxin, T-2HS-BSA antigen was prepared by the method of Succinic Anhydride. After immunizing Balb/c mice with the conjugation for several times,totle RNA was isolated and the VH and VL genes were amplified separately by RT-PCR.The scFv DNA was assembled with a Linker by SOE-PCR. The vector of pCANTAB5E-scFv was constructed and electrotransformed into E.coli TG1 and amplified with the co-infection of help phage M13K07 to obtain phage display library. ELISA was used to select the phage clone displaying scFv of the antibody.After four rounds of panning with T-2HS-KLH,we obtained a phage clone T1 that giving relative strong signal in ELISA.To investigate the effects of T-2 toxin on SP2/0, cells were treated with T-2 toxin (7.8~125 ng/mL) for 0~48h, the morphology of the cells were detected by inverted microscope, the cell viability was evaluated by MTT assay, apoptosis-related gene Caspase-3 was determined by detection the mRNA expression via real time PCR. The results showed that apoptosis induced by T-2 toxin is in a dose and time dependent manner after exposures to T-2 toxin. Meanwhile, T-2 toxin could also up-regulate the expressions of Caspase-3 in a time-dependent manner. These data indicate that Caspase-3 may play an important role in the development of T-2 toxin-induced apoptosis in SP2/0 cells. |
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