类免疫球蛋白（Hemolin）是一种鳞翅目昆虫特有的免疫相关蛋白,也是唯一的无脊椎动物免疫球蛋白家族成员。本实验应用RACE技术获得了茶尺蠖（Ectropis obliqua Prout）类免疫球蛋白基因的全长cDNA序列,命名为EoHML（GenBank登录号：KM885983）,并分析了相关生物信息学特性,检测了病毒感染后基因的表达水平。结果表明,EoHML基因序列全长1β772βbp,包含1β239βbp的开放阅读框,编码412个氨基酸,预测蛋白分子量大小为45.8βkD,等电点（pI）为8.297,属于典型的分泌型蛋白,且具有昆虫Hemolin基因保守的4个Ig功能区和2个N-glycosylation位点。系统进化分析表明,该蛋白与目前已知的类免疫球蛋白氨基酸序列的亲缘关系都较远,与烟草天蛾（Manduca sexta）类免疫球蛋白氨基酸序列相似度最高,为53%。荧光定量检测结果表明,茶尺蠖核型多角体病毒（EoNPV）感染茶尺蠖幼虫后该基因表达量显著升高,最高表达量是正常对照的36.5倍,表明茶尺蠖EoHML基因可能参与了茶尺蠖对EoNPV的免疫代谢反应。
Hemolin, a unique immune protein, belongs to the immunoglobulin superfamily of Lepidoptera insects. A full length cDNA sequence of hemolin gene was cloned by RACE-PCR from tea geometrid (Ectropis obliqua Prout), which was named as EoHML (GenBank Accession No. KM885983). The results of bioinformatics characterization showed that the full length cDNA of EoHML was 1β772βbp, which contained an intact CDS of 1β239βbp and encoded 412 amino acid residues with a putative molecular mass of 45.8βkD and an isoionic point of 8.297. The deduced amino acid sequence demonstrates it belongs to a typical secretory protein that contains 4 Ig functional conservative areas and 2 N-glycosylation sites. Phylogenetic analysis showed that EoHML share the far genetic distance with published hemolin gene and the highest similarity with that of Manduca sexta was only 53%. The results of quantitative real-time PCR analysis further revealed a significant up-regulated expression about 36.5 times in the larvae infected E.obliqua Nucleopolyhedrovirus (EoNPV) than the controls. The study indicated that the EoHML gene may participate in the immune response to EoNPV in tea geometrid.
[1] Faye I, Pye A, Rasmuson T, et al. Insect immunity Ⅱ Simultaneous induction of antibacterial activity and selection synthesis of some hemolymph proteins in diapausing pupae of Hyalophora cecropia and Samia cynthia[J]. Infection and Immunity, 1975, 6(12): 1426-1438.
[2] Sun S, Lindstrom I, Boman H G, et al. Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily[J]. Science, 1990, 250(4988): 1729-1732.
[3] Lee K Y, Horodyski F M, Valaitis A P, et al. Molecular characterization of the insect immune protein hemolin and its high induction during embryonic diapause in the gypsy moth, Lymantria dispar[J]. Insect Biochemistry and Molecular Biology, 2002, 11(32): 1457-1467.
[4] Bao Y, Yamano Y, Morishima I.Induction of hemolin gene expression by bacterial cell wall components in eri-silkworm, Samia cynthia ricini [J]. Comparative Biochemistry and Physiology: Part B, 2007, 146(1): 147-151.
[5] 何芳青, 陈琳, 姚慧鹏, 等. 家蚕类免疫球蛋白(hemolin)基因的克隆及表达特征和抗菌活性研究[J]. 蚕业科学, 2010, 36(1): 40-45.
[6] 姚慧鹏, 吴小锋. 昆虫类免疫球蛋白结构和功能研究进展[J]. 昆虫学报, 2008, 51(1): 530-536.
[7] 张益民, 王学兰, 张世敏, 等. 茶尺蠖核型多角体病毒超微结构的初步研究[J]. 科学通报, 1985(24): 1918-1920.
[8] 殷坤山, 陈华才, 肖强, 等. 茶尺蠖核型多角体病毒制剂的试制与推广应用[J]. 中国病毒学, 2000, 15(S1): 81-84.
[9] 席羽, 殷坤山, 肖强. 不同地理种群茶尺蠖对EoNPV的敏感性差异研究[J]. 茶叶科学, 2011, 31(2): 100-104.
[10] 罗聪, 何新华, 陈虎, 等. 一种高效获取基因5′末端的RACE方法[J]. 植物生理学报, 2011, 47(4): 409-414.
[11] Petersen Tn, B S V H. SignalP 4.0: discriminating signal peptides from transmembrane regions[J]. Nature Methods, 2011, 340(8): 785-786.
[12] Tamura K, Peterson D, Peterson N, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.
[13] Lu Y, Yuan M, Gao X, et al. Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)[J]. PloS ONE, 2013, 8(7): e68059.
[14] Teng XL, Zhang Z, He GL, et al. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects[J]. Journal of Insect Science, 2012, 12(60): 1-17.
[15] Bao Y, Lv Z, Liu Z, et al. Comparative analysis of Bombyx mori nucleopolyhedrovirus responsive genes in fat body and haemocyte of B. mori resistant and susceptible strains[J]. Insect Molecular Biology, 2010, 19(3): 347-358.
[16] 袁志军, 张传溪, 肖强, 等. 茶尺蠖核型多角体病毒(EoNPV)实时荧光定量PCR检测方法的优化及应用[J]. 茶叶科学, 2013, 33(3): 229-236.
[17] Olle Terenius H J R P. Bacterial, but not baculoviral infections stimulate Hemolin expression in noctuid moths[J]. Developmental and Comparative Immunology, 2009(33): 1176-1185.
[18] Hirai M, Terenius O, Li W, et al. Baculovirus and dsRNA induce Hemolin, but no antibacterial activity, in Antheraea pernyi[J]. Insect Molecular Biology, 2004, 13(4): 399-405.
浙公网安备 33019902000101号 
