Multiform single chain variable fragments (scFvs) including different length linker scFvs and bispecific scFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between VH and VL of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON scFv and antizearalenone (anti-ZEN) scFv was also constructed through connection by a short peptide linker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecific scFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgenic plants.
Shihua WangCengjie ZhengYing LiuHuirong ZhengZonghua Wang
In this paper,we developed a rapid and accurate method for the detection of Vibrio parahaemalyticus strains,using multiplex PCR and DNA—DNA hybridization.Multiplex PCR was used to simultaneously amplify three diagnostic genes(tlh,tdh and fla) that serve as molecular markers of V.parahaemolyticus.Biotinylated PCR products were hybridized to primers immobilized on a microarray,and detected by chemiluminesce with avidin—conjugated alkaline phosphatase.With this method,forty-five samples were tested.Eight known virulent strains (tlh^+/tdh^+/fla^+) and four known avirulent strains(tlh^+/tdh^-/fla^+) of the V.parahaemolyticus were successfully detected,and no non-specific hybridization and cross-hybridization reaction were found from fifteen closely-related strains(tlh^-/tdh^-/fla^+) of the Vibrio spp.In addition,all the other eighteen strains of non-Vibrio bacteria(tlh^-/tdh^-/fla^-) gave negative results.The DNA microarray successfully distinguished V. parahaemolyticus from other Vibrio spp.The results demonstrated that this was an efficient and robust method for identifying virulent strains of V.parahaemolyticus.
Rongzhi WangJiadong HuangWei ZhangGuangmei LinJunwei LianLibin JiangHongcong LinSongfa WangShihua Wang
