家蚕模式识别受体βGRP4的序列分析及诱导表达

Sequence Analysis and Induced Expression of the Pattern Recognition Receptor βGRP4 in Silkworm,Bombyx mori

β-葡聚糖识别蛋白(βGRP)属于模式识别受体(PRR),很多无脊椎动物的βGRP具有结合β-1,3-葡聚糖的能力,在识别入侵病原物和激活免疫调控途径中起着重要作用。对克隆的家蚕βGRP4(BmβGRP4,GenBank登录号为:GQ372969)及编码蛋白进行序列分析和诱导表达,探讨其参与免疫应答的作用方式。BmβGRP4的开放阅读框(ORF)长1 128 bp,编码375个氨基酸,具有一个由17个氨基酸组成的信号肽,预测成熟体蛋白分子质量为40.3 kD,等电点为6.5。BmβGRP4含有一个糖苷水解酶活性结构域(Glyco_hydro_16),具有结合β-1,3-葡聚糖的能力。将BmβGRP4亚克隆到p28载体进行原核表达,获得带有His标签的重组蛋白。给家蚕5龄第3天幼虫分别以1×109个/头的剂量添食大肠杆菌、家蚕黑胸败血菌和家蚕白僵菌,对免疫诱导18 h后家蚕体内的BmβGRP4表达水平进行Western blotting分析,结果表明BmβGRP4能被上述3种微生物诱导而产生较强的表达活性。研究结果提示BmβGRP4可能在家蚕先天免疫应答反应中通过识别病原微生物表面的相关分子模式而发挥作用。

β-glucan recognition proteins（βGRPs） belong to pattern recognition receptors（PRR）.Most βGRPs are capable of binding β-1,3-glucan in most invertebrate species,playing important roles in recognizing the intrusive pathogens and activating immunoregulatory pathways.In present study,sequence analysis and induced expression of a cloned Bombyx mori βGRP4 gene（Bm βGRP4,GenBank accession No.GQ372969） were conducted to investigate its functioning mechanism in immune response.It was found that Bm βGRP4 has an open reading frame（ORF） of 1 128 bp and codes for 375 amino acids,with a predicted signal peptide of 17 amino acids.The calculated molecular mass of the mature peptide is about 40.3 kD and the theoretical pI is 6.5.BmβGRP4 contains a structural domain with glycosidic hydrolase activity（Glyco_hydro_16）,being capable of binding β-1,3-glucan.We subcloned Bm βGRP4 into p28 vector for prokaryotic expression and obtained the recombinant protein with the His·Tag.At 18 h after each day 3 larva of the 5th instar was orally infected with 1×109 cells of Escherichia coli,Bacillus bombysepticus and Beauveria bassiana respectively,Western blotting was conducted to determine the expression level of BmβGRP4 in the larva.The results showed that a high level expression of Bm-βGRP4 could be induced by the three kinds of microbe.These results suggest that BmβGRP4 is likely to play roles in Bombyx mori innate immune response through recognizing molecular patterns associated with cell surfaces of the pathogenic microorganisms.