Expression Of ProPO Gene Of Penaeus Monodon

The prophenoloxidase gene of the black tiger shrimp ( Penaurs monodon ) cloned into pET-28a(+) vector was expressed in E.coli BL21. A special band of 79.4 kDa was detected using SDS-PAGE and Western-blotting, which matched the theoretical molecular mass(82.4 kDa) of the fusion protein on the whole. The obtained inclusion body was denatured, purified with Ni-NTA agarose and renatured, resulting in the enzyme activity of 0.3851 unit/mg. The transformed E.coli BL21 were induced under different conditions. The soluble recombinant protein could be detected only when bacteria were induced at 18℃, and the expression ratio came to a head at the 9th hour post-induction, which reached 5.56% of the total soluble protein in E.coli. The soluble recombinant protein purified was digested with trpisin, resulting in the special bands of 60 kDa and 44.5 kDa detected using SDS-PAGE, and the enzyme activity of 0.4249 unit/mg was observed in the solution of digested soluble recombinant protein.To obtain the recombinant plasmid pIZT/V5-His-proPO, the prophenoloxidase gene of the black tiger shrimp, Penaurs monodon, was cloned into pIZT/V5-His vector. Bm-N cell was transfected with pIZT/V5-His-proPO by using liposome. Sieved using antibiotic ZeocinTM, the transfected cells expressing the target proPO stably was gained. A special band of 81.5 kDa, higher than the theoretical molecular mass of the fusion protein(79.4 kDd), was detected by using SDS-PAGE and Western-blotting, confirming that proPO gene was expressed successfully in the transfected Bm-N cell with some modifications.