การแสดงออกและลักษณะสมบัติของ SERINE PROTEINASE HOMOLOGUE (SPH) ในกุ้งกุลาดํา RECOMBINANT EXPRESSION AND CHARACTERIZATION OF SERINE PROTEINASE HOMOLOGUE (SPH) FROM BLACK TIGER SHRIMP

A full-length cDNA of the serine proteinase homologue (SPH) of Penaeus monodon was identified by rapid amplification cDNA end (RACE) method. The complete cDNA sequence of 1,958 bp contains an open reading frame (ORF) of 1,572 bp encoded a 523 amino acid protein including a 19 amino acid signal peptide. The calculated molecular mass of the mature protein is 51.58 kDa with an estimated pI of 4.86. The expression of P. monodon SPH mRNA was determined in the hemocytes of Vibrio harveyi-injected shrimp by in situ hybridization. The results showed the induction of SPH transcript upon V. harveyi challenge. The catalytic domain of P. monodon SPH was expressed in bacteria system. Then, the recombinant protein was purified using the Ni column and S agarose column. The partially purified protein was used for preparation of a specific polyclonal antibody. This antibody will be further used to study SPH distribution in various shrimp tissues using immunohistochemistry. Finally, the function of SPH was investigated by RNA interference (RNAi). The results suggested that the shrimp SPH probably acts as a cofactor of the prophenoloxidase system.