| Encapsulation reaction, an effective and rapid activity to eliminate large quantities of pathogens or parasites in insects, required the participation of granular hemocytes and plasmatocytes and the state of plasmatocytes must be changed from spherical cells into adhesive and flattened cells in encapsulation. Integrins, presented essentially on the surface of cells, are transmembrane glycoproteins that connecting intracellular and extracellular-matrix for signal transduction. Integrin played important roles in development of Drosophlia and innate immune response of some lepidoptera insects. Based on the cloning of Ostrinia furnacalis integrinβ1 (Ofintβ1), the tissue distribution and functions of Ofintβ1 in regulating spreading and encapsulation of hemocytes are studied in Ostrinia furnacalis larvae.For detecting Ofintβ1 protein, a 408 bp Ofintβ1 cDNA fragment closed to 5'terminal was obtained (aa# 174-309), which containing a ligand binding site (aa# 173-177). The fragment Ofintβ1 was ligased with pET-32a and then the positive clone was transmited into E. coli strain BL21(DE3) and was expressed effiently by IPTG induction. SDS-PAGE showed that a 27.6 kDa soluble fusion protein was expressed, which could specifically bind to monoclonal antibody of 6His by Western blot, suggesting a 6His tag exited in the C-terminal of the recombinant protein. The fusion protein was purified with Ni-NTA and then took as the antigen to immunize the New Zealand rabbit to prepare polyclonal antibody against OfINTβ1(Anti-OfINTβ1). Titer of the antiserum was 12 800 detacted with ELISA analysis. Western blot showed that the antiserum could specifically recognize the fusion protein OfINTβ1.Total RNAs were extracted from hemocytes, brains, cuticles, fat bodies, mid-guts, malpighian tubes and tracheae of Ostrinia furnacalis larvae in metaphase of fifth instar. RT-PCR analysis on the transcription level suggested that Ofintβ1 were only detected in hemocytes, but not in other tissues. And Western blotting of different tissues both decteced OfINTβ1 expression in the extraction of hemocytes induced and non-induced. Immunolocaliatioin results showed that Anti-OfINTβ1 could only specifically bind to hemocytes.Antisera diluted in different concentrations were used to block out OfINTβ1 in the surface of hemocytes. Results showed that the adhesion of hemocytes to slide was different by the different concentrations of antisera. After adhesion for 30 min on the slides, 2×diluted antisera treated hemocytes did not congregated and had no any changes in the phase. Furthermore, adhesion of 5×and 10×diluted antisera treated hemocytes to the slide was obviously inhibited, and 100×diluted antisera treated hemocytes almost had no any effects to adhesion of hemocytes in which showed the similar states as in control naive serum and Pringle's saline. Results suggested OfINTβ1 play key roles in the process of state changing of plasmocytes and adhesion of hemocytes.RNA interference (RNAi) was used to analysis the function of Ofintβ1 in encapsulating foreign targets. First, recombinant vector L4440-Ofintβ1 was constructed and expressed in E. coli strain HT115(DE3) to produce Ofintβ1 dsRNA, which were feed on Ostrinia furnacalis larvae. But no interference effect was detected. Then Ofintβ1-dsRNA was synthesized in vitro and microinjected into larva's hemocoel. Some effects of RNAi could be observed after injection in telophase of fourth instar and then ecdysis. Expression of Ofintβ1 decreased and this resulted in the inhibition of encapsulation. These results suggested preliminarily that Ofintβ1 play a key role in the course of encapsulation on foreign objects. |
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