Detection Of Vibrio Parahaemolyticus By Gene Chip And Single Chain Antibody Varible Fragment (scfv)

Vibrio parahaemolyticus, a gram-negative motile bacteria and free-living inhabitant of marine and estuarine environments throughout the world, is a major foo dborne pathogen that causes diarrheain human through seafood consumption. In this study, rapid and accurate methods for detection for Vibrio parahaemolyticus strains were established by using gene chip and fusion protein based on immunoflurescenced scFv.We designed a single multiplex PCR to simultaneously amplify three diagnostic regions(tlh, tdh and FlaB virulent genes). the 367 bp fragment of the tlh gene was amplified by primer tlh1 and tlh2, the 269 bp tdh fragment and 176 bp FlaB fragment were amplified by primer tdh and primer FlaB respectively. DNA-DNA hybridization with specifical PCR primers which were anchored on chip, the result of hybridization were analysised with biotin-avidin alkaline phosphatase reaction. With the proposed method, thirty six samples were tested, eight known virulent strains of Vibrio parahaemolyticu were successfully detected, four isolates of Vibrio parahaemolyticus were identificated as tlh⁺/tdh^-. No extra DNA bands were found from closely related strains of the Vibrio parahaemolyticu. In addition, twenty-four strains of other bacteria were tested using this method, negative results were obtained in all the tests.In order to prepare the scFv, we amplificated tlh gene from Vibrio parahaem olyticus genome by PCR and constructed the fusion expression vector pET32-tlh, expressed the fusion protein in E. coli BL21 by inducing with IPTG.. The thioredoxin were used to increase the soluble yields of the fusion protein and the function of His₆ is to facilitate the purification of expected protein. SDS-PAGE shown that expected protein was about 45% of the total bacterial proteins, the expected protein was purified with Ni²⁺ affinity chromatography.After immunization mice with Trx-TLH, mRNA was isolated and the V_h and V_l were amplified separately by RT-PCR. The scFv DNA fragment was assembled with a Linker. The vector of pCANTAB-5E-scFv was constructed and the vector was transformed into TG1. In order to yield recombinant phages, infected the cell with M13KO7 helper phage. ELISA was used to selected the phage clone displaying scFv fragment of the antibody. After six rounds of panning with Trx-TLH, we obtained a phage clone WW6 that giving strong signal in ELISA.