柞蚕Kazal型丝氨酸蛋白酶抑制剂ApKTSPI基因克隆及表达特征分析
Molecular cloning and expression profiles of a Kazal-type serine protease inhibitor gene in Antheraea pernyi
王 磊** 邱建烽 钱 岑 朱保建 刘朝良
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作者单位：安徽农业大学生命科学学院，合肥 230036
中文关键词：柞蚕，Kazal，基因克隆，表达特征
英文关键词：Antheraea pernyi, Kazal, gene cloning, gene expression
中文摘要：
【目的】 克隆柞蚕（Antheraea pernyi）Kazal型丝氨酸蛋白酶抑制剂（ApKTSPI）基因的cDNA序列并进行序列分析，研究ApKTSPI基因的组织表达分布及病原物免疫刺激后的表达模式，原核表达ApKTSPI。【方法】 利用RACE-PCR方法扩增柞蚕ApKTSPI基因全长cDNA，生物信息学软件进行序列分析，利用实时定量PCR检测柞蚕ApKTSPI基因的组织分布及免疫刺激后的表达模式，利用pET-28a载体在大肠杆菌BL21中融合表达ApKTSPI。【结果】 柞蚕ApKTSPI基因的cDNA全长568 bp，开放阅读框编码96个氨基酸，含一个Kazal结构域。ApKTSPI基因在柞蚕5龄幼虫脂肪体中特异性高表达，在核型多角体病毒、大肠杆菌和白僵菌免疫刺激后表达量都能上调，但上调的程度和时间都不同。ApKTSPI在大肠杆菌中成功诱导表达。【结论】 获得了柞蚕ApKTSPI基因的cDNA全长，并研究了ApKTSPI基因的表达模式，为进一步研究其在柞蚕免疫中的功能及作用机理奠定了基础。
英文摘要：[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.