PI3K和受体型酪氨酸激酶介导家蚕麻痹肽免疫诱导信号的传递

【目的】普遍存在于鳞翅目昆虫中的ENF肽具有非常相似的结构和生理功能, 对昆虫的发育、 免疫、 应激反应等方面都起着重要的调控作用。有研究报道, 作为ENF肽家族成员之一的家蚕Bombyx mori麻痹肽（paralytic peptide, PP）通过激活MAPK来调控家蚕的免疫应答, 但其完整的分子作用机制尚不明确。本研究旨在解析传递家蚕PP免疫诱导信号的分子通路。【方法】首先通过荧光定量PCR检测了多种昆虫细胞系在PP诱导下抗菌肽基因的转录水平, 并利用Western blot检测p38 MAPK的磷酸化水平, 然后利用不同信号传递分子的抑制剂处理BmE细胞筛选参与传递PP免疫诱导信号的分子。【结果】PI3K抑制剂LY294002和受体型酪酸激酶抑制剂Genistein抑制了PP对BmE细胞抗菌肽基因表达的诱导作用; 且BmE细胞和家蚕血细胞在PP的诱导下, Akt和大小约为70 kDa的细胞膜蛋白均出现磷酸化水平的动态变化。【结论】PI3K/Akt信号通路和Genistein敏感性的受体型酪氨酸激酶介导了PP免疫诱导信号的传递。     

中国野蚕一种强抗病毒蛋白的基因分析和活性鉴定

以最近报道的家蚕抗病毒蛋白基因为线索,从中国野蚕（Bombyx mandarina Moore）中肠内克隆了抗家蚕BmNPV病毒的SP-2 cDNA（GenBank登录号：AY945210）,基因大小855bp,编码284个氨基酸的蛋白质,分子量29．6kD,基因组全长1376bp,包含5个外显子和4个内含子．该基因的表达仅限于中肠,具有组织特异性,在幼虫龄中表达水平较高,而在眠期和熟蚕没有表达．推导其氨基酸序列,发现其C端氨基酸序列与已报道的家蚕相应序列差别较大,有8个氨基酸完全不同．通过体外重组技术,由高效基因表达系统获得大量重组蛋白,发现该蛋白具有很强的抗家蚕BmNPV活性,与家蚕对应的抗病毒蛋白BmSP-2相比,其抗BmNPV活性高1．6倍．初步认为,该蛋白质C端序列差异可能是造成家蚕与野蚕抗病毒活性差别的主要原因．     

家蚕神经肽基因的筛查及成熟肽的预测

神经肽（neuropeptide）是家蚕Bombyx mori体内重要的调控物质。为了充分理解神经肽对家蚕发育的调控, 扩充家蚕神经肽的数量, 本研究利用BLAST的tblastn程序结合OpenOffice软件的查找程序, 基于其他昆虫和无脊椎动物神经肽的同源性和保守的结构特点, 在家蚕基因、理论蛋白质数据库及NCBI中进行全面而系统的基因筛查; 并利用各种在线工具对所筛查到基因和理论蛋白质的结构和成熟肽进行预测分析。结果共获得allatostatin-A （AST-A）, allatostatin-B （AST-B）, allatostatin-C （AST-C）, allatropin (AT), ecdysis-triggering hormone （ETH）, crustacean cardioactive peptide (CCAP)和FMRFamide等31个神经肽基因家族, 包括37个神经肽基因亚家族, 共计44个神经肽基因; 预测出193个成熟的神经肽, 其中73个根据家族同源性预测在C末端发生酰胺化, 而6个被预测在N末端发生了环化, 9个被预测酪氨酸发生了硫酸化。大部分成熟神经肽都具有明显的家族结构特征, 但proctolin, CCAP及CAPA-PK成熟肽结构上与其他昆虫相比有所扩展。结果提示, 家蚕神经和内分泌细胞产生了几乎在所有昆虫中具有的神经肽前体; 进化过程中大部分成熟神经肽的氨基酸序列在种间产生了差异, 但家族特征性基序高度保守。本研究为神经肽功能研究以及神经肽对家蚕发育尤其是蛹期发育调控的研究奠定了基础。     

家蚕胚胎细胞系Bm-Em-1的建立和特性研究

昆虫细胞系在病毒生物学、 基因功能的研究以及杆状病毒表达系统生产重组蛋白的应用中发挥着重要的作用。本研究由家蚕Bombyx mori “大造”品种反转期胚胎建立了一株细胞系Bm-Em-1, 在含10％胎牛血清的TNM-FH培养基中已传代40余代。显微观察表明, 细胞形态主要为圆形和短梭形, 细胞染色体呈短棒状和颗粒状, 数量多、 异倍化, 符合典型的鳞翅目昆虫细胞染色体特征。RAPD鉴定结果表明, 该细胞系来源于家蚕胚胎, 其扩增谱带与BTI-Tn5B1-4和Sf-9等细胞系明显不同。生长曲线测定结果表明, 第28代细胞的群体倍增时间为82.2 h。病毒敏感性测定显示, 该细胞系不能被苜蓿银纹夜蛾Autographa californica核型多角体病毒（AcMNPV）感染, 但对家蚕核型多角体病毒（BmNPV）高度敏感, 96 h的感染率为91.3％。结果说明该细胞系可作为家蚕病毒离体复制、 BmNPV表达系统以及家蚕基因功能研究的理想材料。     

家蚕核型多角体病毒egt基因的分子进化分析

过PCR方法获得家蚕核型多角体病毒（Bombyx mori nuclearpolyhedrosis virus,BmNPV）的蜕皮甾体尿苷二磷酸葡萄糖基转移酶基因（egt）片段,序列分析表明该片段带有EGT的完整ORF,推测的多肽可形成EGT结构域的高级结构。为了研究egt的起源,利用家蚕基因组数据库,电子克隆了多个家蚕尿苷二磷酸葡萄糖醛酸转移酶（UGT）基因,在此基础上进行了进化分析,表明BmNPV的EGT为antennal-enriched型UGT;推测核型多角体病毒（nucleopolyhedrivirus,NPV）和颗粒体病毒（granulovirus,GV）的egt基因在进化上来源于昆虫的UGT基因,但GV的egt基因在进化上的起源可能要早于NPV的egt基因;可能在昆虫祖先种进化形成不同昆虫目的某一时期,杆状病毒的祖先种从昆虫中获得了antennal-enriched型UGT基因,并进化为egt基因。家蚕的部分UGT基因与转座子元件连锁的基因组结构特点反映了杆状病毒的egt基因可能通过转座子的传递而获得。     

家蚕核型多角体病毒水平转移基因分析

为了探讨杆状病毒基因组的遗传进化模式,文章利用家蚕核型多角体病毒(BmNPV)和其宿主家蚕全基因组数据,进行了全基因组的同源性搜索和系统进化分析,结果显示,BmNPV的几丁质酶(Chi)基因、凋亡抑制蛋白3(IAP3)基因和尿苷二磷酸葡萄糖转移酶(UGT)基因为水平转移基因。这3个基因都来源于其宿主昆虫。通过核苷酸组成、密码子偏好性、选择压力等基因特征分析,发现BmNPV水平转移基因与其基因组序列存在明显差异,进一步验证水平转移基因的外源性。对3个水平转移基因的功能分析发现它们有利于杆状病毒在宿主昆虫中的侵染与繁殖,并提高杆状病毒在昆虫中的生存能力。     

BmNPV和AcNPV扩大寄主域杂交重组病毒表达载体的构建和改进

苜蓿银纹夜蛾核型多角体病毒(Autographa californica nuclear polyhedrosis virus, 简称AcNPV)和家蚕核型多角体病毒(Bombyx mori nuclear polyhedrosis virus, 简称BmNPV)表达系统是目前最主要的两类昆虫杆状病毒表达系统. 两者各具特点, 前者寄主昆虫个体较小, 但寄主范围广、适合较大规模的细胞悬浮培养生产体系; 而后者虽然寄主专一, 但以个体较大、易饲养的家蚕为寄主, 具有表达量高、 特别适合产业化水平开发的要求. 为了利用AcNPV和BmNPV各自的优势, 扩大昆虫杆状病毒的寄主范围, 以BmNPV为基础, 利用决定病毒寄主范围的DNA解旋酶基因及同源重组原理, 构建了介于BmNPV和AcNPV间的杂交重组病毒(hybrid baculovirus of AcNPV and BmNPV, 简称HyNPV). 这样构建的杂交重组病毒具有AcNPV和BmNPV的双重优点, 既能感染所有AcNPV的寄主, 又可以在家蚕中表达. 以人碱性成纤维细胞成长因子基因作为应用实例, 克隆构建了这样的一种重组杂交病毒, 可在Sf21, Tn-5, BmN培养细胞及家蚕个体中穿梭表达. 为了减少表达后重组蛋白的降解, 利用“基因敲除”法删除了杆状病毒本身的一种主要蛋白酶-半胱氨酸蛋白酶基因, 提高了重组蛋白的稳定性和表达效率.     

氯霉素乙酰基转移酶基因在家蚕核型多角体病毒P10基因启动子控制下的表达

昆虫核型多角体病毒(NPV)P10基因属于晚期基因,为强启动子所控制,又是病毒复制所非必需的基因。我们对前报的家蚕核型多角体病毒(BmNPV)P10基因,应用PCR技术进行ATG区定点突变,在ATG被突变的同时形成一个BglⅡ酶切位点,得到一个不含ATG的BmNPV P10基因启动子。将长为230bp的经突变后的BmNPV P10基因5’端(包括启动子所有特征)片段克隆进pMMTV·CAT质粒中,构建成一个CAT基因在BmNPV P10基因启动子控制下的pBmPl0·CAT瞬间表达质粒。该质粒通过转染进入经野生型BmNPV感染的BmN细胞中,CAT得以表达。证明BmNPV P10启动子是比较强的启动子,可以在BmN细胞表达外源基因,具备了作为表达载体启动子的特性。     

Bac-to-Bac/BmNPV杆状病毒在家蚕幼虫中表达重组鲎C因子

鲎C因子是鲎血细胞中一种对内毒素具有高亲和力的丝氨酸蛋白酶,被内毒素激活后可水解人工合成的三肽底物,因而能替代传统的鲎试剂用于内毒素检测。通过RT-PCR从东方鲎血细胞中扩增出鲎C因子基因(factor C)序列,利用Bac-to-Bac/BmNPV杆状病毒表达系统在家蚕幼虫中表达该蛋白,并对其进行活性测定。结果显示:被感染的家蚕血清稀释后能检测到Factor C活性,稀释500倍的血清可以用于内毒素检测,且其灵敏度能达到0.2 EU/mL,有望开发新型的、低成本的内毒素检测试剂。     

hsc70-4基因促进家蚕核型多角体病毒复制增殖

【目的】热休克应答(heatshockresponse,HSR)是机体细胞应对环境压力的一种重要防御策略,鉴定热休克蛋白在杆状病毒侵染宿主过程中的功能,并揭示其作用的分子机制,为探明宿主与病毒相互作用的分子基础提供理论依据。【方法】通过分子克隆技术对Bmhsc70-4基因进行克隆,并利用BioEdit及GeneDoc对其进行多序列比对分析;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对Bmhsc70-4基因进行过表达和敲除;利用荧光定量PCR技术检测相应基因的表达量;通过对Caspase-9和Caspase-3/7活性的检测确定Bmhsc70-4基因对细胞凋亡的影响;通过免疫荧光验证BmHSC70-4和BmIAP的共定位情况,并进一步通过免疫共沉淀验证它们的相互作用。【结果】Bmhsc70-4基因开放阅读框为1950bp,编码649个氨基酸,在昆虫间具有较高的保守性;BmNPV能够诱导Bmhsc70-4基因上调表达,过表达Bmhsc70-4基因能够促进BmNPV的增殖,敲除Bmhsc70-4基因能够抑制BmNPV的增殖,表明Bmhsc70-4基因的表达利于BmNPV的增殖;Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能;荧光共定位显示BmHSC70-4和BmIAP共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;BmNPV侵染过程中Bmhsc70-4基因能够促进Bmiap基因的表达。【结论】Bmhsc70-4基因具有抑制家蚕细胞凋亡的功能,在BmNPV侵染家蚕细胞过程中,能够与BmIAP相互作用,并促进BmNPV复制增殖。     

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.     

利用荧光差异显示技术分离的家蚕抗NPV相关基因s3a

通过荧光差异显示技术,分析了家蚕Bombyx mori对BmNPV抗性品系NB、感性品系306和近等基因系306NNZZ添毒和未添毒处理区的基因表达的差异。根据差异显示的结果克隆了一条702 bp长度的cDNA片段,并用Northern blot进行了验证。该序列经过NCBI EST库的同源性比较获得了电子延伸。延伸后的序列用特异引物进行RT-PCR扩增获得了一条782 bp的序列,拼接后基因cDNA序列全长为827 bp,推导的氨基酸序列与草地夜蛾Spodoptera frugiperda S3A同源性最高达97.7％;其次是烟芽夜蛾Heliothis virescens S3A,同源性为94.0％;与黑腹果蝇Drosophila melanogaster S3A同源性为75.3%。比较结果显示这是一个新的家蚕基因,定名为家蚕s3a基因。本实验获得的s3a基因在家蚕感性和抗性品系以及添毒处理和未添毒处理中都具有差异表达,其中在抗性品系和近等基因系中的表达高于感性品系,在添毒处理中的表达高于未添毒组。因此推测它是一个与家蚕抗BmNPV相关的新基因。     

Identification and characterization of an NPV infection-related gene Bmsop2 in Bombyx mori L.

Abstract:  Using the fluorescent differential display technique, we analysed the differential expression of genes related to Bombyx mori nuclear polyhedrosis virus (BmNPV) resistance. Silkworm strains studied included the highly resistant strain NB, highly susceptible strain 306 and near-isogenic line 306NNZZ. One novel gene was identified and named Bmsop2 for its high similarity with the Sop2 protein of other species. It was identified to be linked to BmNPV susceptibility by Northern blotting and real-time polymerase chain reaction. The results indicated that it was actively expressed in midguts of strains 306, NB and the eighth generation of backcross (BC₈) of strain 306NNZZ which had been treated with BmNPV. But the expression level was low in the midguts of the control. In the mean time, the expression of Bmsop2 was the highest in strain 306 treated with BmNPV while it was the lowest in strain 306 not treated with BmNPV. Our study showed that Bmsop2 is a differentially expressed gene in strains NB, 306 and 306NNZZ which have different levels of resistance to BmNPV.     

Enhanced production of secretory beta1,3-N-acetylglucosaminyltransferase 2 fusion protein into hemolymph of Bombyx mori larvae using recombinant BmNPV bacmid integrated signal sequence

The enhanced secretion of beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) fusion protein into the hemolymph of Bombyx mori larvae was studied using a recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid integrating seven signal sequences. When the BmNPV bacmid encoding the signal sequences from the silkworm B. mori bombyxin (bx) and B. mori prophenoloxidase-activating enzyme (ppae) was injected into silkworm larvae, 56.1 and 51.5mU/ml beta3GnT, respectively, were secreted into the hemolymph of silkworm larvae. For bx, 97.3% of the total beta3GnT activity was secreted into hemolymph, and only 1.1% remained in the intestines of silkworm larvae. For ppae, 90.8% of the total beta3GnT activity was secreted to the hemolymph, but 7.8% remained in the intestines of silkworm larvae. Using the BmNPV bacmid encoding bx, the amount of secreted beta3GnT was 91mug per larva, which was 2.5% of the total amount of protein in the hemolymph.     

Comparative proteomic analysis reveals that caspase-1 and serine protease may be involved in silkworm resistance to Bombyx mori nuclear polyhedrosis virus

The silkworm Bombyx mori is of great economic value. The B. mori nuclear polyhedrosis virus (BmNPV) is one of the most common and severe pathogens for silkworm. Although certain immune mechanisms exist in silkworms, most silkworms are still susceptible to BmNPV infection. Interestingly, BmNPV infection resistance in some silkworm strains is varied and naturally existing. We have previously established a silkworm strain NB by genetic cross, which is highly resistant to BmNPV invasion. To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we employed proteomic approach and genetic cross to globally identify proteins differentially expressed in parental silkworms NB and 306, a BmNPV-susceptible strain, and their F(1) hybrids. In all, 53 different proteins were found in direct cross group (NB♀, 306♂, F(1) hybrid) and 21 in reciprocal cross group (306♀, NB♂, F(1) hybrid). Gene ontology and KEGG pathway analyses showed that most of these different proteins are located in cytoplasm and are involved in many important metabolisms. Caspase-1 and serine protease expressed only in BmNPV-resistant silkworms, but not in BmNPV-susceptible silkworms, which was further confirmed by Western blot. Taken together, our data suggests that both caspase-1 and serine protease play a critical role in silkworm resistance against BmNPV infection.     

Isolation and structure determination of a paralytic peptide from the hemolymph of the silkworm, Bombyx mori.

A peptide with paralytic activity in larvae of the silkworm, Bombyx mori, was isolated from its hemolymph. Purification procedures consisted of extraction with 50% acetone, Vydac C4 reversed-phase cartridge elution and 4 steps of reversed-phase HPLC. Injection of the purified peptide into 4th instar B. mori larvae caused rapid and rigid paralysis for 2 min at a dose of 3.4 ng/larva. This paralytic peptide consists of 23 amino acid residues containing 2 cysteines with an intra-disulfide bond. The complete amino acid sequence is: H-Glu-AsnPhe-Val-Gly-Gly-Cys-Ala-Thr-Gly-Phe-Lys-Arg-Thr-Ala-Asp-G ly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The relationship between structure and the biologic activity of synthetic analogs indicated that the entire amino acid sequence and the intra-disulfide bond were necessary for biological activity.     

Cloning and partial characterization of a gene in Bombyx mori homologous to a human adiponectin receptor

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.     

Arginine kinase is highly expressed in a resistant strain of silkworm (Bombyx mori, Lepidoptera): Implication of its role in resistance to Bombyx mori nucleopolyhedrovirus

A gene encoding Bombyx mori arginine kinase (BmAK) has been indentified differentially expressed in the midguts of Bombyx mori strain NB which is resistant to nucleopolyhedrovirus (BmNPV), strain 306 which is susceptible to NPV and a near isogenic line BC(8) with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). In this study, we characterized the expression profiles of BmAK using RT-PCR and real-time quantitative PCR. The expression level of BmAK fluctuated in various developing stage and various tissue. Remarkably, the expression level of BmAK increased more than 10-fold 24 hours post inoculation (h p.i.) of NPV in strain NB and BC(8), while such increment was abraded in strain 306 although the basal expression level of BmAK in strain 306 was higher than that of strain NB and BC(8). Western blotting analysis using polyclonal antibody against BmAK verified such observation, and immunofluoresence analysis indicated for the first time that BmAK was mainly located to the cytoplasm or some structures in cytoplasm. These findings suggest that arginine kinase is involved in the antiviral process of Bombyx mori larvae against NPV infection.