Construction of eukaryotic recombinant expression plasmids with glyceraldehydes-3-phosphate dehydrogenase and cysteine protease inhibitor gene of periodic Brugia malayi and its expression in HeLa cells

Abstract

Objective To construct the eukaryotic expression plasmid containing glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and cysteine protease inhibitor ( CPI ) gene from periodic Brugia malayi (Bm) and to lay foundation for studying multivalent vaccines. Methods Total RNA was extracted from periodic Bin. The BmGAPDH and BmCPI genes were amplified by RT-PCR. The PCR product was cloned and then subeloned into eukaryotic recombinant plasmid vector pcDNA3.1 (+). pcDNA3.1 (+)/BmGAPDH/BmCPI was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into HeLa cell subsequently. The transient expression of BmGAPDH and BmCPI were examined by RT-PCR. The expressed protein was identified by sodium dodeeylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE). Results Two specific bands of around 877 bp of BmGAPDH and 621 bp of BmCPI were amplified, consistent with the expected value. The same bands were obtained by double restriction enzyme digestion of recombinant plasmids or PCR using recombinant plasmid as template. BmGAPDH and BmCPI mRNA were highly expressed in transfeeted HeLa cell. The relative molecular mass (Mr) of the recombinant protein was about 54 × 103. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/BmGAPDH/BmCPI has been constructed successfully and the protein is expressed correctly in mammalian cell. Key words: Brugia malayi; Glyceraldehydes-3-phosphate dehydrogenases; Cysteine proteinase inhibitors; Recombination, genetic; HeLa cells