Clone Of PTV Swine/CH/IMH/03 3' End Sequence And Expression And Immunogenocity Research Of VP1 Protein

Porcine teschovirus (PTV) was described as causative agents of severe and mild neurological disorders known as Teschen/Talfan disease, reproductive failure, pneumonia, diarrhea, and dermal lesions of swine. Nowadays Talfan/Teschen disease was worldwide, and had brought great economic loss. Therefore, study of PTV vaccine, diagnosis and immunologic mechanism had been concerned with by more and more researches.3'end sequence of Picornaviruses had 100-500 base pairs. Studies had shown that 3'end sequence of Picornaviruses were relation to infection and replication of virus. Teschovirus and foot and mouth disease virus were Picornaviridae. Scientists had researched clearly 3'end sequence of FMDV, but structure and function of 3'end sequence of teschovirus were not clear. According to SWINE/CH/IMH/03 sequence of PTV published in Genebank and utilizing RACE (Rapid Amplification of cDNA Ends) technology, we gained complete 3' end sequence of the SWINE/CH/IMH/03 strain of porcine teschovirus. The study could be helpful to research structure and function of genome.According to SWINE/CH/IMH/03 sequence of PTV published in Genebank, one pair of primers were designed, and approximate 804bp of VP1 gene was amplified using RT-PCR. By restrictive enzyme digestion of EcoR I and Xho I, VP1 gen and pET-32a(+) vector are both restrictived, then utilizing T4 DNA ligase VP1 gen was directed positively cloned to pET-32a(+) vector. Recombiant plasmids were transformed BL21(DE3), which were induced by IPTG (final concentration 1.0mmol/ml) at 37℃for 6 hours. About 45kDa protein was detected with SDS-PAGE, and the objective protein quantity was up to 63.4%. VP1 recombinant protein could be recongnized by anti-PTV serum.VP1 recombinant protein was purified with GE His GraviTrap^TM affinity columns. Purified protein by SDS-PAGE electrophoresis and thin-layer scanning showed that purity of VP1 protein reached 97%. Then using purified protein injected 5 BALB/C mice that were 7-8-weeks old. Mice were thrice immunized, then blood was collected, and sera were extracted. IFA, ELISA and Western Blot test demonstrated that VP1 recombinant protein possessed good immunogenicity. Antibodies to VP1 recombinant protein had neutrality activety. That was to say, this express protein could be used as candidate for diagnostic antigen and recombinant subunit vaccine.