Cloning, Expression And Immunogenicity Analysis Of T24 Gene From Cysticercus Cellulosae

Total RNA was extracted from Cysticercus cellulosae, the T24 gene specific primers were designed using Oligo software. A 716 bp specific fragment was amplified by RT-PCR and ligated into pGEM-T Easy vector. It was identified by restriction endonuclease analysis, PCR and sequencing that the fragment contained the complete open reading frame (ORF) of the T24 gene. The homologies of the nucleotide sequence and encoding amino acid sequence were 98% and 100% respectively compared with the sequence published in Genebank.The recombinant plasmid pGEM-T24 was digested with EcoR I and SalI, releasing the T24 gene fragment. The fragment subsequently was inserted into the EcoR I and Not I sites of the prokaryotic expression vector pGEX-4T-1 and was transformed into E.coli BL21, this construct was also sequenced to ensure fidelity. Expression of the recombinant plasmid was induced by IPTG in E. coli resulting in a high level of protein expression. The expressed products were analyzed by SDS-PAGE and Western-blotting. The results of SDS-PAGE showed that the 40 ku T24 GST fusion was expressed successfully. Western-blotting showed good immunoreactivity of the expressed product. The expressed product was used to immunize mice. The antisera were collected from the immunized mice and examined by ELISA. The results showed that immunization of mice generated antibodies and the antibodies specifically reacted with T24.The recombinant plasmid pGEM-T24 was digested with EcoR I and Not I, releasing the T24 gene fragment. The fragment subsequently was inserted into the EcoR I and Not I sites of the eukaryotic expression vector pPIC9K and was transformed into E.coli JM109. Positive recom- binants named pPIC9K-T24 was linearized by SalI, then the linearized DNA was transformed into P·pastoris GS115 by electroporation. MD was used to select HIS+ transformants and then utilized hyper-resistance to G418 to screen for possible multicopy strains. PCR analysis showed that the gene of interest was integrated within the genome of the mulicopy recombinants. The recombinants were induced to express products by methanol. The cultures supernatant was collected and tested by SDS-PAGE and Western-blotting. It showed that the T24 gene was expressed successfully in P·pastoris and the molecular weight was about 21 ku. The 21 ku protein can be recognized by the positive serum from patient cysticercosis.This study cloned T24 gene from Cysticercus cellulosae successfully and expressed in prokaryotic and eukaryotic respectivly. The expressed products confirmed good immunogenicity. It provided theoretic proof and material for developing genetic engineering vaccine.