家蚕C类清道夫受体BmSR-C的基因克隆、原核表达及亚细胞定位

doi:10.16380/j.kcxb.2018.10.002

摘要/Abstract

摘要： 【目的】本研究旨在克隆与鉴定家蚕Bombyx mori C类清道夫受体基因BmSR-C，为探析其在家蚕免疫中的功能奠定基础。【方法】利用RACE技术克隆家蚕C类清道夫受体基因全长序列，并对其进行生物信息学分析。利用RT-PCR和qPCR方法对BmSR-C基因的时空表达情况进行检测。通过原核表达和Ni⁺亲和层析的方法获得BmSR-C重组蛋白，免疫昆明小鼠制备抗BmSR-C多克隆抗体。利用ELISA法和Western blot分别对鼠抗BmSR-C蛋白多克隆抗体的效价和特异性进行检测。构建家蚕BmSR-C的真核表达载体，转染家蚕BmE细胞，分析该蛋白的亚细胞定位情况。【结果】克隆获得家蚕BmSR-C基因(GenBank登录号: BGIBMGA004577)，其开放阅读框(ORF)全长为1 821 bp，编码606个氨基酸残基。BmSR-C具有典型的C类清道夫受体家族结构特征，主要由CCP, MAM和SO结构域以及靠近C端的单次跨膜结构域组成。进化分析结果显示鳞翅目昆虫SR-C单独聚为一支，家蚕BmSR-C与同为鳞翅目昆虫的草地贪夜蛾Spodoptera frugiperda和大红斑蝶Danaus plexippus的同源蛋白亲缘关系最为接近。对BmSR-C基因的时空表达分析表明，BmSR-C在家蚕的马氏管和血细胞中高表达，而在其他组织中无明显表达；其在家蚕不同发育时期的血细胞中均有表达，且在4龄眠期的表达量达到峰值。ELISA检测结果显示，所制得抗体效价高达 1∶128 000；Western blot检测结果显示，该抗体可以特异性识别重组蛋白。家蚕BmE细胞中的亚细胞定位结果表明BmSR-C主要定位于细胞膜。【结论】获得家蚕C类清道夫受体基因BmSR-C的完整cDNA序列及其表达特征；成功制备了BmSR-C的多克隆抗体，利用家蚕BmE细胞在细胞水平上分析了BmSR-C的亚细胞定位情况，推测其参与家蚕的生长发育及病原微生物入侵的免疫反应，为进一步研究BmSR-C的生物学功能奠定了基础。

关键词: 家蚕, BmSR-C, 表达分析, 原核表达, 亚细胞定位

Abstract: 【Aim】 This study aims to clone and identify a class C scavenger receptor gene BmSR-C in the silkworm, Bombyx mori, so as to lay a foundation for the further exploration of the function of scavenger receptor class C in the silkworm immunity. 【Methods】 The full-length cDNA of BmSR-C was cloned by RACE and analyzed by bioinformatics. The spatial and temporal expression profiles of BmSR-C were detected by RT-PCR and qPCR. The BmSR-C recombinant protein was obtained by prokaryotic expression and Ni⁺ affinity chromatography. The antibody was acquired by using the recombinant protein to immunize mice, and its titer and specificity were detected by ELISA and Western blot, respectively. Simultaneously, the subcellular localization of the protein was analyzed after BmSR-C eukaryotic vector was constructed and then transfected into BmE cells of the silkworm. 【Results】 We cloned the fulllength cDNA of BmSR-C (GenBank accession no.: BGIBMGA004577) from B. mori, which contains an ORF of 1 821 bp encoding a putative protein of 606 amino acid residues. BmSR-C has the typical structural features of scavenger receptor class C family, including CCP, MAM and SO domains, as well as a single transmembrane region in the near end of the C-terminus. Evolutionary analysis showed that SR-C proteins from lepidopteran insects clustered alone, and BmSR-C was most closely related to its homologues from Spodoptera frugiperda and Danaus plexippus. The spatial and temporal expression profiles showed that BmSR-C was highly expressed in the Malpighian tubules and hemocytes, but showed no obvious expression in other detected tissues. In addition, BmSR-C was expressed in hemocytes during different developmental stages, with the expression peak at the 4th instar larval molting. The titer of the antibody was estimated as high as 1∶128 000 dilution ratio through ELISA, and the result of Western blot showed that the antibody could specifically recognize the recombinant protein. The subcellular localization result in BmE cells showed that BmSRC is mainly located on the cell membrane. 【Conclusion】 BmSR-C was cloned and its expression patterns were investigated. The antimouse polyclonal antibody was obtained. The subcellular localization of BmSR-C was investigated in BmE cells of the silkworm. It is inferred that BmSR-C may be involved in the growth and development and immune response to the invasion of pathogenic microorganisms in the silkworm. This study provides a foundation for further research of the biological function of BmSR-C in the silkworm.

Key words: Bombyx mori, BmSR-C, expression profiling, prokaryotic expression, subcellular localization

申利, 许川, 张蕾, 张奎, 毛景欣, 潘光照, 李重阳, 胡仁建, 杨丽群, 崔红娟. 家蚕C类清道夫受体BmSR-C的基因克隆、原核表达及亚细胞定位[J]. , 2018, 61(10): 1132-1144.

SHEN Li, XU Chuan, ZHANG Lei, ZHANG Kui, MAO Jing-Xin, PAN Guang-Zhao, LI Zhong-Yang, HU Ren-Jian, YANG Li-Qun, CUI Hong-Juan. Gene cloning, prokaryotic expression and subcelluar localization of a class C scavenger receptor BmSR-C in the silkworm, Bombyx mori [J]. , 2018, 61(10): 1132-1144.

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链接本文: http://www.insect.org.cn/CN/10.16380/j.kcxb.2018.10.002

http://www.insect.org.cn/CN/Y2018/V61/I10/1132

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家蚕C类清道夫受体BmSR-C的基因克隆、原核表达及亚细胞定位

Gene cloning, prokaryotic expression and subcelluar localization of a class C scavenger receptor BmSR-C in the silkworm, Bombyx mori

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