Research On Immune Suppressive Protein On The Surface Coat Of Steinernema Glaseri

Entomopathogenic nematodes are widely used as biological control agents which can suppress or evade the host immune defense upon entry into insects. Immune suppressive protein from Steinernema glaseri surface coat was proved to play important roles in defeating the host immune system. For the purpose of studying the mechanism and application of immune suppressive protein, we purified the immune suppressive protein from S. glaseri surface coat, cloned gene of the protein and performed expression and localization. Mechanism of immune suppressive protein was preliminarily studied and application of the protein was performed.The main results are as summarized as follows:1. Gene of immune suppressive protein was cloned.A protein fraction with antiphagocytosis activity was separated and further analyzed with two-dimensional electrophoresis (2-DE). Mass spectrometry analysis of protein spot from2-DE gel gave peptides showing high similarity with enolases of many organisms. Degenerative primers were designed based on conserved region of enolases. Coding region of immune suppressive protein was then cloned from infective juveniles by using RT-PCR and RACE. The amino acid sequence of immune suppressive protein showed high homology to those of enolases from other organisms and had features of enolase family. The protein was designated as Sg-ENOL.2. Sg-ENOL expression and antibody production.Sg-ENOL was successfully expressed in E. coli by using pGEX-6p-1vector. GST-tag of the recombinant protein was removed by enzyme digestion. After several steps of purification, non-tagged Sg-ENOL was obtained. Conversion of2-PGE to PEP was catalyzed by recombinant Sg-ENOL, indicating the protein is an enzymatically active enolase. A polyclonal antibody against Sg-ENOL was produced and purified.3. Localization of Sg-ENOL and preliminarily study on mechanism of immune suppressive protein.Immuno-gold TEM analysis confirmed the location of Sg-ENOL on nematode surface and muscle cells. After incubation with insect homogenate other than PBS, S. glaseri secreted Sg-ENOL to medium. Secretion of Sg-ENOL to insect hemolymph by S. glaseri revealed that Sg-ENOL was related to infection.Sg-ENOL was expressed in insect cells by constructing recombinant baculovirus. After expression, recombinant Sg-ENOL was retained in cytosol instead of exported to medium which indicated a specific secretion mechanism in S. glaseri. Specific binding property of Sg-ENOL to plasminogen shed light on investigation of insect interactive protein of Sg-ENOL.4. Sg-enol gene was transformed to Metarhizium and recombinant Metarhizium with high virulence was obtained.Injection of Sg-ENOL to Locusta migratoria manilensis Meyen enhanced virulence of Metarhizium. By using a gene gun system, Sg-enol gene connected with pbar-EX vector was transformed to Metarhizium. The transformants were verified by PCR. Insect bioassay revealed that virulence of transformants was enhanced, as indicated by significantly shorten survival curve of Locusta migratoria manilensis Meyen and lower50%lethal times (LT50) of the transformants compared to the values for the wild-type strain. These results fully demonstrated the application potential of Sg-enol gene in biological control.