飞蝗几丁质酶5-1抗体制备及表达特性分析

doi:10.3864/j.issn.0578-1752.2018.12.018

Abstract

Abstract: 【Objective】 The objective to this study is to prepare polyclonal antibody of Locusta migratoria chitinase5-1 (LmCht5-1), establish method for detection of LmCht5-1 protein, understand the expression pattern of LmCht5-1 protein in the 4thinstar nymph and finally confirm its location and function in vivo.【Method】LmCht5-1 antigen structure area, which was selected by sequence alignment between LmCht5-1 and LmCht5-2 with MEGA software and antigen epitope prediction analysis with the website of Expression, was amplified using cDNA of L. migratoria as the template. The LmCht5-1 antigen sequence was cloned into expression vector pET32a-OVA, which contains ovalbumin to improve immunogenicity, to generate the antigen expression recombinant plasmid pET32a-OVA-LmCht5-1. pET32a-OVA-LmCht5-1 was then transformed into E. coli strain BL21 (DE3). The IPTG induced OVA-LmCht5-1 was purified through Ni-NTA system and used as the antigen to generate polyclone antibody by rabbit immunization. The titer and specificity of OVA-LmCht5-1 were detected by ELISA and Western blot analysis, respectively. Then the expression pattern of LmCht5-1 in cuticle of the 4th instar nymph was analyzed in parallel by Western blot. Finally, the intracellular localization and function of LmCht5-1 in the 4thinstar nymph cuticle were detected by immunohistochemical analysis after the dsLmCht5-1 injection. 【Result】The 5′ fragment (471-533AA) of LmCht5-1, which was selected as the antigen area with the sequence alignment between LmCht5-1 and LmCht5-2 and the epitope prediction analysis, was inserted into pET32a-OVA to obtain recombinant plasmid pET32a-OVA-LmCht5-1. The 71.34 kD recombinant protein OVA-LmCht5-1 was expressed, purified and immunized with rabbit to generate polyclone antibody. The titer of anti-serum of OVA-LmCht5-1 was 1﹕102 400 by ELISA detection. Antibody OVA-LmCht5-1 could accurately distinguish the LmCht5-1 but not LmCht5-2 in Western blot analysis. Further, it was found that the protein expression level of LmCht5-1 increased gradually with the age in the 4th instar nymph of L. migratoria and reached to the highest at the same day of molting, while it declined to the lowest after molting. Injection dsLmCht5-1 into the 4th instar nymph led to the expression of LmCht5-1 significantly declined to 70.0% in mRNA level and 73.6% in protein level. The immunohistochemical analysis with the epidermis of L. migratoria after dsLmCht5-1 injection, showed that LmCht5-1 was located in old cuticle and epidermis cell. Finally, it was found that the inhibition of protein expression of LmCht5-1 led to the chitin degradation blocking in old cuticle. 【Conclusion】 High titer and specificity of LmCht5-1 antibodies, which could be used for Western blot and immunohistochemistry analysis were obtained. The protein expression of LmCht5-1 reached to the highest level at the day of molting in 4th instar nymph. LmCht5-1 was located in the epidermis cell and old cuticle in the 4th instar nymph. Moreover, the injection of dsLmCht5-1 to L. migratoria could inhibit the protein expression of LmCht5-1 in epidermis cells and old cuticle, and finally caused the chitin degradation deficiency in old cuticle.

Key words: Locusta migratoria, LmCht5-1, polyclonal antibody preparation, expression pattern analysis, Western blot

ZHANG TingTing, LIU WeiWei, GAO Lu, LI RenJian, FU SuiYe, LIU XiaoJian, LI DaQi, LIU WeiMin, DONG Qing, ZHANG JianZhen. The antibody preparation and expression analysis of Chitinase 5-1 in Locusta migratoria [J].Scientia Agricultura Sinica, 2018, 51(12): 2418-2428.

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The antibody preparation and expression analysis of Chitinase 5-1 in Locusta migratoria

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