家蚕C-型凝集素11基因BmCTL11的克隆及表达分析

Cloning and Expression Analysis of C-type Lectin 11 Gene in Bombyx mori

昆虫的C-型凝集素参与先天免疫系统中细胞间的相互作用,家蚕C-型凝集素11(C-type lectins,CTL11)基因BmCTL11是经家蚕微孢子虫(Nb)感染诱导后在蚕体内持续高量表达的基因。用生物信息学方法分析BmCTL11基因编码有307个氨基酸的多肽,预测分子质量34.51 kD,等电点4.69,含2个串联的典型C-型凝集素糖识别结构域,与烟草天蛾的IML-1蛋白进化距离最近。RT-PCR检测BmCTL11基因在家蚕幼虫时期的转录水平较高,并在多个组织有转录。将灭活的家蚕病原细菌、真菌、微孢子虫、病毒等注射家蚕5龄幼虫后的24 h内,qRT-PCR检测BmCTL11基因在幼虫脂肪体和中肠组织内的转录水平均发生上调,说明该基因能够被多种病原微生物诱导。以家蚕5龄第3天幼虫脂肪体c DNA为模板克隆了BmCTL11基因,连接到p ET-32a载体,诱导表达后将纯化的融合蛋白免疫昆明鼠制备出效价为1∶102 400的BmCTL11多克隆抗体。Western blot分析结果显示,BmCTL11蛋白可能以二聚体形式主要分布于家蚕脂肪体、血浆、中肠、表皮等组织中,推测BmCTL11在家蚕识别病原微生物并介导引发进一步的先天免疫反应过程中有重要的作用。

C-type lectin involves in the cellular interaction in innate immune of insect. Bombyx mori C-type lectin 11 gene（BmC TL11） was expressed continuously at high level after infection with Nosema bombycis（Nb）. Bioinformatic analysis revealed that BmC TL11 encodes a peptide of 307 amino acid with molecular weight of 34. 51 kD and isoelectric point of4. 69. The deduced protein contains two tandem typical C-type lectin carbohydrate recognition domains and has the minimus genetic distance with Manduca sexta IML-1 protein. RT-PCR revealed that the transcriptional level of BmC TL11 gene was high at larval stage, and could be detected in various tissues of silkworm larvae. Within 24 h after injected inactivated silkworm pathogenic bacteria, fungus,Nb and viruses into the 5th instar silkworm larvae,qRT-PCR showed that the transcriptional level of BmCTL11 gene was increased in fat body and midgut,indicating that BmC TL11 can be induced by different pathogenic microorganisms. BmC TL11 was amplified using c DNA of fat body from the 5th instar silkworm larvae as template and subsequently cloned into p ET-32 a plasmid. The recombinant plasmid was transformed into E. coli Rosetta（ DE3） for induced expression of BmC TL11 protein. Mouse polyclonal antiserum whose titer was over 1∶ 102 400 was collected after fusion protein BmC TL11 was purified and injected into Kunming mice. Western blot analysis revealed that BmC TL11 mainly distributed in fat body,plasma,midgut and epidermis of silkworm in a form of dimers. These results suggested that BmC TL11 may play an important role in pathogen recognition and innate immune responses.