Identification And Immunological Function Of Small Serpin Gene In Nasonia Vitripennis

Nasonia vitripennis is an important natural enemy of many species of flies.This study was conducted to identify and characterize an exclusive serine protease inhibitor from N.vitripennis venom,and to explore its mechanism of inhibiting the host immune system.Parasitic wasps have been used as biological control agents instead of chemical control which causes environmental pollution,thus to find out use of N.vitripennis as a good biological agent is important.Humoral response is one of important modulators of innate immune system of insects.Humoral immunity plays a vital role by producing antibacterial peptides,antiviral factors,protease inhibitors and melanogenesis.Serine protease inhibitors,as regulatory factors of enzyme activity in organisms,participate in the blackening reaction caused by PPO pathway,and thus play a role in protecting the host.Small Pacifastin protease inhibitor(SPPI),a type of serine protease inhibitor,widely regulates a variety of biological processes,but its role in immune regulation of parasitic wasps is poorly understood.In this experiment,SPPI gene of parasitic wasp was cloned,and its immunological function and mechanism of action were explored.The main results were as follows:1.Clone and identify the parasitic wasp NvSPPI cDNA sequence,the length of ORF is 243 bp,and encode 81 amino acid residues.The n-terminal contains a signal peptide composed of 24 amino acids,which is a secretory protein.The predicted molecular weight of protein was 8.61 kDa and the isoelectric point was 7.56.The analysis of evolutionary tree shows that the Pacifastin light chain domains(PLDs)of the parasitic wasp NvSPPI is closely related to the Ceratosolen solmsi marchali.2.The fusion protein with a molecular weight of about 32 kDa was induced to express in E.coli.The soluble pGEX-4T-2-NvSPPI recombinant protein with high concentration and purity was obtained by Ni-NTA affinity chromatography.3.NvSPPI was expressed in the head,chest,abdomen,ovarian tissue and venous tissue of parasitic wasps,of which the highest expression was found in the venous tissue and the difference was significant.NvSPPI was almost not expressed on 0 and 1 days post eclosion and reached the highest level on 4 days post eclosion,and then decreased gradually.4.Arylphorin subunit A4 interacting with NvSPPI was preliminarily screened out from the non-denatured hemolymph protein of Musca domestica by pull-down technique.5.The effect of the recombinant protein NvSPPI on the activation of the hemolymph itself phenol oxidase(PO)and proPO in houseflies was tested.The results showed that the recombinant protein had significant inhibitory effect on the activation of the hemolymph prophenol oxidase of the housefly,but could not affect the phenol oxidase activity of the hemolymph itself.6.The enzyme inhibition spectrum of NvSPPI protein was determined.The results showed that the recombinant GST-NvSPPI protein had inhibitory effect on trypsin,but had no inhibitory effect on chymotrypsin and protease K.