| Classical Swine Fever(CSF) is one of major contagious diseases in pig breeding industry. In order to gain massive diagnostic antigen CSFV, and large scale of CSFV specific major protective antigen protein(E2 envelope glycoprotein) in eucaryotic cells, which are prepared into subunit vaccine for effective prevention against CSF. E2 gene from CSFV HL-LY isolate was cloned and expressed in Pichia Pastoris expression system, moreover, conditions were explorerd for expressing the E2 envelope glycoprotein. The results are showed as follows.According to CSFV's E2 gene sequence published in GenBank and the sequence of P.Pastoris expression vector pPIC9's Multiple Cloning Site(MCS), a pair of primers were designed with Oligo and PrimerS.O softwares. E2 gene was amplified by RT-PCR from HL-LY isolate. The product of RT-PCR was cloned to pMD18-T vector, and identified by restriction endonuclease, PCR and sequencing, which was named as T-E2.The E2 gene's sequence was analyzed by homology comparing with published E2 gene of CSFV Alfort strain, Brescia strain, SHIMEN strain, C strain, C-V-LZ strain, GS-LT strain and GS-LX strain.The homology of E2 gene complete sequence shares 99.20%, 95.44%, 96.51%, 95.53%, 89.12%, 78.66% and 78.23% in nucleotide respectively and shares 98.93%, 95.98%, 97.32%, 95.71%, 89.54%, 83.16% and 83.42% identities in amino acid sequence respectively.The major antigenic region of the sequence was also analyzed by homology comparing, which shares 98.90%, 95.05%, 96.70%, 96.15%, 95.42%, 82.42%, and 83.15% in nucleotide respectively and shares 98.35%, 95.05%, 97.25%, 95.60%, 95.05%, 87.36% and 88.46% identities in amino acid sequence respectively.The result shows that E2 gene is higher conservative, but there exist CSFV E2 gene mutation strains in China according to its lower homologies with C-V-LZ , GS-LT and GS-LT strains which were isolated from the different regions in China.E2 gene was cut with restriction endonuclease from the recombination plasmid T-E2, and subcloned into the expression vector pPIC9's MCS, which was identified by enzyme digestion and PCR amplification. The recombination expression vector, pPIC9-E2, was linearized by Sal I and electroporated into P.Pastoris GS115. Recombinant P.Pastoris GS115, designated GS115-pPIC9-E2, was identified by PCR and the product of the PCR was analyzed by sequencing before its expression for CSFV E2 protein.Expression of CSFV E2 fusion protein was followed in Recombinant P.Pastoris GS115-pPIC9-E2 with the induction of 1% methanol at 30 C, shaking at 200rpm. A strong protein band of molecular mass estimation 50-54ku with SDS-PAGE was seen. This protein was detected on Western blots and Dot-ELISA probed with anti-CSFV serum, but P.Pastoris GS115 without recombinant E2 showed no protein in this region. It was estimated that theyield of the E2 fusion protein in culture supernatant of Recombinant P.Pastoris could be reached 0.34g/L.The optimal expression conditions were explored for the pH, aeration rate, final concentration of methanol and kinds of growth media. We conclude that the higher quantity of E2 protein can be gained at pH4.0-6.0, 1% methanol as the final concentration, MM growth media and high aeration rate.Candidate:Man Chaolai Speciality:Preventive Veterinary Science Supervisor:Prof. Li Yijing... |
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