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家蚕C型凝集素S11的克隆、 表达及功能研究
Cloning, expression and analysis of the silkworm C-type lectin S11
詹明月** 杨佩瑾 饶相君***
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DOI:10.7679/j.issn.2095-1353.2017.095
作者单位:安徽农业大学植物保护学院,合肥 230036
中文关键词:家蚕,先天免疫,C型凝集素,免疫受体,凝集
英文关键词:silkworm, innate immunity, C-type lectin, immune receptor, agglutination
中文摘要:

【目的】 C型凝集素(C-type lectin, CTL)广泛存在于植物和动物中,参与免疫防御反应、发育及细胞信号传导。鳞翅目昆虫中的C型凝集素依据长度和特征功能域可以分成S型、IML型和X型,目前S型凝集素的功能了解非常有限。本研究对家蚕Bombyx mori L. C型凝集素S11进行了克隆、蛋白表达纯化和初步功能分析。【方法】 基于先前对家蚕基因组中所有C型凝集素序列的生物信息学分析,通过PCR克隆了CTL-S11基因的全长cDNA;利用相关软件对核酸和蛋白质序列进行了比对和分析;通过大肠杆菌原核系统表达纯化重组蛋白;通过RT-PCR和荧光定量PCR研究了该基因的组织表达分布和诱导表达;通过凝集实验检测了重组蛋白对细菌的凝集作用。【结果】 获得了长度为519 bpCTL-S11全长cDNA序列,编码173个氨基酸,纯化得到分子量约为18 ku,带有组氨酸标签的重组蛋白。RT-PCR表明该基因在中肠、脂肪体、血细胞和表皮都有表达。荧光定量PCR结果表明,细菌喂食或注射家蚕幼虫后,CTL-S11的表达水平在某些时间点有显著上调。凝集实验显示,在钙离子存在的条件下重组蛋白对大肠杆菌和金黄葡萄球菌有明显的凝集作用。【结论】 CTL-S11的表达可以被细菌显著诱导,重组蛋白可以引起细菌的凝集,因此其有可能作为免疫受体参与家蚕对病原微生物的识别。

英文摘要:

 [Objectives]  C-type lectins are common in plants and animals and are involved in immune defense responses, development and signal transductions. Lepidopteran C-type lectins can be divided into three types (S, IML and X) based on their lengths and conserved domains. Little is known about the functions of S-type CTLs. We cloned the Bombyx mori L. C-type lectin S11, quantified its expression, and conducted a preliminary analysis of its function. [Methods]  Based on bioinformatic analysis of all C-type lectin sequences in the silkworm genome, we cloned the full-length cDNA of CTL-S11 with PCR. The nucleic acid and amino acid sequences were compared and analyzed in silico. The recombinant protein was expressed and purified from E. coli. RT-PCR and real-time PCR was used to analyze the tissue profile and inductions. Bacterial agglutination using a recombinant protein was also examined. [Results]  The full-length cDNA of CTL-S11 is 519 bp and encodes 173 amino acids. The corresponding recombinant protein with the HisTag is about 18 ku. RT-PCR indicated that CTL-S11 is expressed in the midgut, fat body, hemocytes and epidermis. Realtime PCR showed that, after feeding, or injecting larvae with bacteria, CTL-S11 was significantly induced at certain time points. The agglutination experiment showed that the recombinant protein induced bacterial agglutination in the presence of Ca2+[Conclusion]  The expression of CTL-S11 was significantly induced by bacteria and a recombinant CTL-S11 protein could induce bacterial agglutination. Therefore, CTL-S11 may function as an immune receptor involved in pathogen recognition in the silkworm.

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