关键词: 飞蝗, 表皮蛋白, LmKnk3-5′, 抗体制备, 组织定位

Abstract:

【Objective】LmKnickkopf3-5′ (LmKnk3-5′) is an important cuticular protein involved in the development of Locusta migratoria. The purpose of this paper is to get the anti-LmKnk3-5′ polyclonal antibody and confirm the localization of LmKnk3-5′ in L. migratoria. The results will be beneficial to the biological function analysis of LmKnk3-5′ from protein level, meanwhile, it can lay the foundation for further study of the interaction of cuticular proteins in cuticle formation of L. migratoria. 【Method】Three specific antigen sequences (R1, R2 and R3) of LmKnk3-5′ were chosen after amino acid sequences alignment of Knickkopf (Knk) family genes in L. migratoria, including LmKnk, LmKnk2, LmKnk3-FL and LmKnk3-5′. The target antigen sequences were amplified by PCR using primers with BamH I, Hind III restriction sites and full-length cDNA sequences of LmKnk3-5′ as template. The antigen sequences and pET-32a vector were all digested by BamH I, Hind III and ligated to each other by T4 ligase enzyme to make recombinant plasmids, then the recombinant plasmids were transformed into BL21 (DE3) competent cells. The cells were incubated with 0.5 mmol·L^-1 IPTG at 16℃ for 20 h to produce the recombinant fusion protein, SDS-PAGE was used to analysis the expression of target proteins. After that, E. coli cells that can express target proteins in dissolved state were expanded cultured for protein extraction. Ni-NTA agarose was used for target proteins purification and BCA method was used to determine the protein concentration. The LmKnk3-5′ polyclonal antibodies were obtained after immunizing BALB/c mouse four times. ELISA and Western blot were used to analyze the antibody titer and specificity, respectively. Finally, the paraffin sections were prepared using the integument of 8-day-old 5th-instar nymphs after dsLmKnk3-5′ and dsGFP injection, immunofluorescence was conducted to confirm the subcellular localization of LmKnk3-5′ in L. migratoria. 【Result】 R1, R2 and R3 were selected as specific antigen regions through amino acid sequences alignment. R1, R2 and R3 contain 208, 147 and 131 aa, respectively, with the predicted molecular weights of 24.0, 17.0 and 14.8 kD, respectively. Three recombinant plasmids (pET-32a-R1, pET-32a-R2, pET-32a-R3) were obtained successfully after enzyme digestion and ligation. SDS-PAGE analysis showed that only the cells consist of pET-32a-R2 plasmids could express target proteins in dissolved state after induced by IPTG. R2 recombinant fusion protein was purified and used to obtain anti-LmKnk3-5′ polyclonal antibody successfully after immunizing mice. ELISA analysis indicated that the titer of LmKnk3-5′ antibody was up to 1﹕512 000. The results of Western blot demonstrated that after dsLmKnk3-5′ injection, the expression of LmKnk3-5′ was significantly decreased in comparison of the dsGFP injection group. The results of immunofluorescence showed that LmKnk3-5′ was located in the epidermal cells and new cuticle, especially the apical site of newly synthesized exocuticle in L. migratoria. 【Conclusion】Anti-LmKnk3-5′ polyclonal antibody was obtained successfully, it has high titer and specificity. LmKnk3-5′ is mainly located in the apical site of newly synthesized exocuticle in L. migratoria. The results will provide protein level evidence for the functional research of LmKnk3-5′ in the cuticle formation of L. migratoria.

Key words: Locusta migratoria, cuticular protein, LmKnk3-5′, antibody preparation, subcellular localization