Abstract: 【Objective】The dsRNA degrading enzyme (dsRNase) is an important obstacle of using RNAi technology for pest control. The objective of this study is to obtain specific antigens of LmdsRNase2 and LmdsRNase3 by using prokaryotic expression system, and then to prepare antibodies, and to detect the protein expression level of midgut and subcelluar localization. The results will provide strong evidence from protein level to further analysis of functional differentiation of LmdsRNase2 and LmdsRNase3 in the midgut of Locusta migratoria.【Method】The specific antigen sequences (R2’ and R3’) of LmdsRNase2 and LmdsRNase3 (GenBank: ARW74135.1, ARW74134.1) were chosen by alignment analysis. The primers with BamHI, HindIII restriction sites were designed according to the complete cDNA sequences. The target sequences were amplified by PCR and ligated to pET-32a vector through double enzyme digestion. The recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells, and then the cells were induced with 0.5 mmol·L^-1 IPTG for 4 hours at 37℃. SDS-PAGE electrophoresis was performed to examine the target proteins. After that, large amounts of E. coli cells were cultured for protein extraction. The target proteins R2’ and R3’ were purified through Ni-NTA agarose chromatography, and the protein concentration was determined according to the method of Bradford. The anti-LmdsRNase2 and 3 polyclonal antibodies were obtained after immunizing New Zealand white rabbit. The specificity of antibody was detected by western blot analysis, the recombinant LmdsRNase2 and LmdsRNase3 proteins were used as antigens. The titers of two antibodies were determined by ELISA. Furthermore, the total proteins were extracted from L. migratoria midgut and midgut fluid on day 3 of 5th instar nymph, and western blot was used to test the expression level of LmdsRNase2 and LmdsRNase3. Finally, the paraffin sections of the midgut on day 3 of 5th instar nymph were prepared, the subcellular localization of LmdsRNase2 and LmdsRNase3 proteins in the midgut of L. migratoria was conducted by immunofluorescence.【Result】LmdsRNase2 and LmdsRNase3 showed 37% sequence identity by full-length amino acid sequence alignment, and the variant sequence regions (named as R2’ and R3’ for LmdsRNase2 and LmdsRNase3, respectively) were selected for PCR primer design. The length of R2’ and R3’ is 157 and 153 a.a., respectively, and the calculated molecular mass of them is 17.0 and 16.8 kD, respectively. The protein expression was induced with 0.5 mmol·L^-1 IPTG for 4 hours at 37℃, finally the expressed proteins were only detected in inclusion bodies. The recombinant proteins R2’ and R3’ were purified using Ni-NTA agarose chromatography, and the purity of R2’ is 85%, it is suitable to directly immune New Zealand white rabbit. But the purity of R3’ is under 85%. To further obtain highly purified R3’, R3’was loaded on a gel and the expected band was cut for protein extraction and following immunization. After 36 days, ELISA results indicated that the titer of LmdsRNase2 and LmdsRNase3 antibodies was 1﹕102 400. Western blot demonstrated that anti-LmsRNase2 and 3 polyclonal antibodies could specifically detect LmdsRNase2 or LmdsRNase3 proteins, respectively, no cross hybridization was observed. The molecular weight was consistent with the predicted size. LmdsRNase2 protein was highly expressed in L. migratoria midgut by western blot, nevertheless, there was no detectable signal in midgut fluid. However, LmdsRNase3 could not be detected in both midgut and midgut fluid. Immunofluorescence results showed that both LmdsRNase2 and LmdsRNase3 were expressed in cytoplasm of L. migratoria midgut cells, but the expression level of LmdsRNase3 protein was much lower than that of LmdsRNase2.【Conclusion】Anti-LmdsRNase2 and 3 specific polyclonal antibodies were successfully prepared, western blot and immunofluorescence showed that LmdsRNase2 protein was highly expressed in midgut, whereas the expression of LmdsRNase3 was very low. These results provide evidence of protein level for the functional differentiation of LmdsRNases in the midgut of L. migratoria.

Key words: Locusta migratoria, dsRNA degrading enzyme (dsRNase), polyclonal antibody, subcelluar localization