Molecular Cloning And Expression Profiles Of A Kazal-type Serine Protease Inhibitor Gene In Antheraea Pernyi

Serine proteinase inhibitors are a superfamily of proteins, which control protease-mediated processes by inhibiting their cognate serine protease. Serine proteinase inhibitors have been reported to involve in some biological procession, such as blood coagulation, fibrinolysis, inflammatory response, cell differentiation and apoptosis, tissue reconstruction and immune reaction. Serine proteinase inhibitors can be classified into some types, Hinudim family, Stefins family, Cystatins family, Cysteine proteinase inhibitors family, and Squash seed inhibitors family.In this study, we cloned a full-length cDNA of a Kazal-type serine proteinase inhibitor in Antheraea pernyi (ApKTSPI) by RACE-PCR method. The full-length cDNA of ApKTSPI was 568 nucleotides long and contained a 291 nucleotide ORF encoding a 96-residue amino acid sequence. Multiple sequence comparison alignment with other Kazal-type serine proteinase inhibitor sequences showed that ApKTSPI has high identity with lepidopteran Kazal-type serine proteinase inhibitor. The phylogenetic analysis result indicated that ApKTSPI clustered with Antheraea mylitta in a grou To analyse ApKTSPI expression level in native tissue by real time PCR, ApKTSPI gene was especially high expressed in fat bodies of 5th larval. After NPV, E. coli or Beauveria bassiana immune-challenged, ApKTSPI gene in fat bodies was up-regulated. However, the up-regulated expression degree and time of ApKTSPI gene were different by three pathogens immune-challenged. To get it express in Escherichia coli, ApKTSPI gene was inserted into pET-28a vector. Recombinant plasm id was transformed into E. coli BL21, and ApKTSPI protein was expressed with IPTG induction. The recombinant ApKTSPI protein was successfully expressed in E. coli.The ApKTSPI gene was successfully cloned and expressed in E. coli, and ApKTSPI may be involved in innate immune reactions against pathogens infection.