丽蝇蛹集金小蜂毒液丝氨酸蛋白酶抑制因子抑制寄主免疫的作用机理

Nasonia vitripennis, as a model organism of hymenoptera and an important natural enemy resource of insects, the deep research has been executed on its individual biology, while hardly on the interaction with its host Musca domestica. In our early research, we found the venom of Nasonia vitripennis could regulate the transcriptional level of immune genes from hemocytes of Musca domestica pupae, and analyzed the composition of venom protein combining the transcriptome and proteomics methords. Based on these, we plan to clone the full length cDNAs of venom serpins and carry out the prokaryotic expression to ascertain the transcription and expression patterns of serpins. Meanwhile, we intend to execute the eukaryotic expression of venom serpins to clarify their substrates spectrum and biological function, then screen the target proteins of its host by yeast two-hybrid system and analyse the expression patterns of positive proteins. Ultimately, we’ll clarify the interaction mechanism of how venom serpins of Nasonia vitripennis inhibit the immune response of its host. This will not only provide the theoretical basis for relationship between parasitoids and its host and reveal the mechanism of insect innate immune response, but also explore new strategies of biological pest control based on inhibition of innate immune response.

现今已有相当多有关丽蝇蛹集金小蜂个体生物学的研究，但其与寄主家蝇互作的研究甚少。本项目在我们前期确定丽蝇蛹集金小蜂毒液能调控家蝇蛹血细胞免疫基因转录的基础上，结合转录组和蛋白质组学，明确了其毒液蛋白组成后，拟采用基因克隆与表达、酶学及功能活性测定和酵母双杂交蛋白质互作等技术，克隆其毒液丝氨酸蛋白酶抑制因子（serpins）的基因全长cDNAs，并进行原核表达，探明serpins基因于不同时期和不同组织的转录与表达模式；同时对serpins进行真核表达，明确其底物谱及生物功能，并筛选寄主中与其互作的靶蛋白，对阳性靶蛋白进行表达模式分析，最终，解析丽蝇蛹集金小蜂毒液serpins抑制寄主免疫反应的作用机理。这将为明确寄生蜂与寄主的相互作用关系和揭示昆虫先天免疫反应的机理奠定理论基础。同时，也为探寻基于抑制先天免疫反应的新型害虫生物防治策略提供新方法。

现今已有相当多有关丽蝇蛹集金小蜂个体生物学的研究，但其与寄主家蝇互作的研究甚少。本项目结合我们前期对丽蝇蛹集金小蜂毒液转录组和蛋白质组分析，明确了其毒液蛋白组成后，采用基因克隆与表达、酶学及功能活性测定、GST pull-down和LC-MS/MS等技术，解析了丽蝇蛹集金小蜂毒液丝氨酸蛋白酶抑制因子serpins抑制寄主免疫的作用机理。共克隆了4个不同类型的丽蝇蛹集金小蜂毒液serpins [NvKSPI-1（kazal型）、NvKSPI-2（kazal型）、NvSPPI（pacifastin型）和NvTIL2（trypsin Inhibitor型）] 的基因全长cDNAs，并进行了原核表达和表达模式分析，结果表明，相对于其它所有组织，4个serpins均于毒液中特异性高表达，是毒液的特异蛋白。酶学及功能活性测定实验表明，4个毒液serpins的底物谱不同，NvKSPI-1、NvSPPI和NvTIL2分别可显著抑制胰蛋白酶、胰蛋白酶和糜蛋白酶的活性；但4个毒液serpins均可通过抑制寄主家蝇蛹血淋巴酚氧化酶原（PPO）的激活反应，来抑制寄主的黑化反应，从而抑制寄主的体液免疫，有助于其成功寄生。GST pull-down及LC-MS/MS分析表明，4个毒液serpins均可与家蝇蛹Arylphorin subunit A4蛋白互作，进一步对寄主的发育和免疫反应进行调控，获得成功寄生。作为膜翅目昆虫的理想模式生物，本项目对丽蝇蛹集金小蜂其毒液与寄主互作机理的解析，一方面，为揭示其它蛹期寄生蜂与寄主害虫免疫反应间的关系奠定理论基础；同时，也为探寻基于抑制先天免疫反应的新型害虫生物防治策略提供新方法。