Molecular cloning and expression profiles of a Kazal-type serine protease inhibitor gene in Antheraea pernyi
Author of the article:WANG Lei** QIU Jian-Feng QIAN Cen ZHU Bao-Jian LIU Chao-Liang
Author's Workplace：College of life science, Anhui Agricultural University, Hefei 230036, China
Key Words：Antheraea pernyi, Kazal, gene cloning, gene expression
Abstract：[Objectives] To clone full-length cDNA of a Kazal-type serine proteinase inhibitor in Antheraea pernyi (ApKTSPI), to analyze its expression level in tissue and in fat bodies after a pathogen’s immune-challenge, and to get it expressed in Escherichia coli. [Methods] ApKTSPI cDNA was amplified using the RACE-PCR method and the expression pattern analyzed using quantitative real time PCR. The ApKTSPI gene was inserted into the pET-28a vector. Recombinant plasmids were transfected into E. coli BL21, and the ApKTSPI protein was expressed with IPTG induction. [Results] The full-length cDNA of ApKTSPI was 568 nucleotides long and contained a 291 nucleotide ORF encoding a 96-residue amino acid sequence. The ApKTSPI gene was especially high expressed in fat bodies of the 5th larval instar. The ApKTSPI gene in fat bodies was up-regulated after challenging the immune system with NPV, E. coli or Beauveria bassiana. However, the degree and timing of up-regulation were different for each of the three pathogens. The recombinant ApKTSPI protein was successfully expressed in E. coli. [Conclusion] The ApKTSPI gene was successfully cloned and expressed in E. coli, and ApKTSPI may be involved in innate immune reactions against pathogens.