家蚕瘫痪肽结合蛋白基因克隆与分析

ENF肽家族具有保守的N末端结构（Glu—Asn—Phe-）。该家族成员肽大多具有重叠功能活性，在鳞翅目昆虫的免疫反应，生长调控和自体调节等方面都发挥着重要的作用。在昆虫的免疫反应中，血细胞尤其是淋巴液的黏附性是针对外来侵入物的免疫应答过程中的重要因素。家蚕瘫痪肽（paralytic peptide）是ENF肽家族的一种，其具有多种的生物学活性，包括致瘫痪性及在家蚕血细胞免疫反应中的促吞噬细胞扩散活性。ENF肽家族的另一成员，粘虫（Pseudaletia separata）的生长阻抑肽（Growth-blocking peptide），同家蚕瘫痪肽一样能够在粘虫的血细胞免疫反应中起到调节吞噬细胞的功能。目前，关于昆虫细胞免疫应答的终端调控分子机制的研究还比较少，有文献报道粘虫的生长阻抑肽结合蛋白（GBP—BP）能够起到沉默生长阻抑肽活性的功能，从而可能参与调节细胞免疫应答的终端调控。在本研究中，利用荧光差异显示技术（FDD）分析了家蚕感染BmNPV病毒后基因表达差异情况，在血淋巴中获得了一条差异条带G12782＊通过5＇-RACE技术，首次在家蚕中克隆得到了该基因的全长cDNA序列。通过同源性分析得知，该基因所编码的蛋白质与粘虫的生长阻抑肽结合蛋白具有很大的同源性，并被命名为家蚕瘫痪肽结合蛋白（Bmori paralytic peptide binding protein，PP-BP）。通过RT-PCR研究发现，该蛋白基因在血淋巴中大量表达。同时，利用实时荧光定量PCR（Real-time quantitative PCR）技术分析了该基因在正常饲养家蚕与添食BmNPV病毒的家蚕中的表达差异，结果显示该基因在家蚕添食BmNPV病毒后的表达量大大增强，这就暗示该基因可能与BmNPV病毒刺激后所引起的家蚕血液细胞免疫反应相关。利用生物信息学方法对该基因的结构进行了分析，发现该基因具有两个外显子和一个内含子。这个基因已经登入GenBank数据库，收入号为DQ306881。

Cloning and Characterization of Bombyx mori PP-BP,a Gene Induced by Viral Infection

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP).Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12782) was obtained from the hemolymph RNA pools. Using 5'-RACE with a specially designed primer based on the FDD study,a 1 401 bp cDNA clone was obtained containing a 1 311 bp open reading frame (ORF, GenBank accession number DQ306881).The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.