家蚕抗核型多角体病毒病育种研究进展

家蚕核型多角体病毒(Bombyx mori nuclear polyhydrosis virus,BmNPV)病是蚕业界上最常见、危害比较严重的病害;遗传学研究和抗性分析表明,家蚕对核型多角体病毒抗性由主效基因和微效基因协同控制。在蚕病防治方面,除了加强消毒以外,选育抗病毒新品种无疑是更加经济有效的办法。近年来,随着基因组学和新一代测序技术的迅猛发展,覆盖深度达8.5倍家蚕基因组图谱完成和40个家蚕品种和野生种重测序的完成,为家蚕的抗核型多角体病毒病育种提供了理论依据和基因资源。本文综述了BmNPV基因组研究、家蚕基因组研究以及家蚕抗性品种培育方面取得的进展。     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

Biochemical analysis of Hyphantria cunea NPV attachment to Spodoptera frugiperda 21 cells

Binding characteristics of Hyphantria cunea nuclear polyhedrosis virus (HcNPV) to Spodoptera frugiperda 21 (Sf21) cells was determined. The cells displayed an affinity of 0.9 × 10¹⁰ M^-1 with about 8900 binding sites per cell. The biochemical nature of HcNPV-binding sites on the cell surface was also partially elucidated. There were 45 to 49% reductions in HcNPV binding following the pretreatment of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, which inhibits N-linked glycosylation and the expression of some membrane proteins on the cell surface, reduced virus binding suggesting a role for glycoprotein(s) in binding. Treatment of cells with wheat germ agglutinin or neuraminidase did not measurably reduce virus binding, indicating that oligosaccharides containing N-acetylglucosamine or sialic acid are not directly involved in HcNPV attachment. The negative effect of methylamine on HcNPV binding seems to be due to the fact that HcNPV entry via an endocytic pathway is blocked by the increased pH of the endosome. Data on energy inhibitors (sodium azide and dinitrophenol) indicates that HcNPV attachment to Sf21 cells may be closely linked to viral entry via receptor-mediated endocytosis. These findings suggest that the binding site moiety has a glycoprotein component, but that direct involvement of oligosacccharides containing N-acetylglucosamine or sialic acid residues in binding is unlikely, and that HcNPV attachment to Sf21 cells might be via receptor-mediated endocytosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

家蚕中肠组织抗核型多角体病毒病的相关蛋白分析

家蚕中肠上皮是病毒经口侵入遇到的第一个组织。昆虫幼虫抵御杆状病毒的感染,可通过选择性的使感染的中肠上皮细胞发生调亡并在释放病毒粒子进入血淋巴之前使感染的细胞从中肠脱落。为研究家蚕抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)病的机制,通过对BmNPV高度抗性和高度敏感性的家蚕品系杂交和回交构建了近等基因系。本文对家蚕高抗,敏感及近等基因系5龄起蚕中肠组织的蛋白质表达谱进行了二维电泳 (two-dimensional gel electrophoresis,2-DE) 分析,并利用基质辅助激光解吸电离飞行时间 (matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 质谱对差异蛋白进行鉴定。结果发现了5个差异表达的蛋白。推测这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。     

Proteomic analysis of differentially expressed proteins involved in BmNPV resistance in the fat body of silkworm, Bombyx mori

To investigate the mechanism of nucleopolyhedrovirus resistance of silkworm, we bred a near-isogenic silkworm line, designated BC9, from the parental resistant strain NB and the susceptible strain 306, that is resistant to infection by nucleopolyhedrovirus. Proteomic techniques were employed to search for candidate genes playing a role in the antivirus response, based on differential protein expression profiles in the fat bodies of these strains. Four proteins were identified, two of which are possibly related to energy metabolism, the third one may have a function similar to integrase, and the fourth one is completely novel. Thus, our strategy of the combined use of near-isogenic silkworm line and proteomic techniques is effective for discovering new genes in the antivirus response of insects.     

Screening of Bombyx mori brain proteins interacting with protein tyrosine phosphatase of BmNPV

In this study, glutathione-S-transferase pull-down combined with mass spectrometry techniques were used to identify the candidate proteins interacting with protein tyrosine phosphatase of the Bombyx Mori nucleopolyhedrovirus in the B. mori (BmNPV-PTP) brain. A total of 36 proteins were identified from BmNPV-PTP coprecipitate samples by searching the NCBI_Bombyx Mori database with the original mass spectrum data. Among those proteins, the interaction between BmNPV-PTP and B. mori cyclophilin A may accelerate the apoptosis of certain nerve cells involved in regulating behavior, and thus may be an inducer of enhanced locomotor activity (ELA). After the BmNPV invasion, BmNPV-PTP binding to peripheral-type benzodiazepine receptors may initiate a series of abnormal cascades of the nervous system, which results in abnormal hyperactive behavior in B. mori. Besides this, vacuolar ATP synthase catalytic subunit A, annexin, and several enzymes for energy conversion were identified, which may play a role in enhancing viral entry and infectivity and provide energy for enhancing the locomotor activity of B. mori. In general, the results of this study will facilitate the understanding of the molecular mechanisms underlying the ELA of B. mori larva induced by BmNPV.     

家蚕生存素基因在BmN4细胞有丝分裂中的功能分析

【目的】阐明生存素基因在家蚕Bombyx mori BmN4细胞有丝分裂中的功能。【方法】qRT PCR分析家蚕生存素基因BmSurvivin在家蚕5龄第3天幼虫不同组织(丝腺、中肠、马氏管、精巢、卵巢、脂肪体、皮细胞层和表皮)中的表达量;构建pIZT/V5-His-BmSurvivin-GFP (BG)融合载体并转染BmN4细胞,以免疫荧光标记检测BmSurvivin和第10位丝氨酸(Ser10)磷酸化的组蛋白H3(H3Ser10ph)在BmN4细胞有丝分裂分裂不同时期的定位。【结果】BmSurvivin在家蚕5龄第3天幼虫马氏管中表达量最高,其次是在丝腺和中肠中。成功构建pIZT/V5-His-BmSurvivin-GFP载体。免疫荧光结果显示,在BmN4细胞间期核中可以看到明显的GFP信号,涵盖了细胞核和细胞质区域,指示BmSurvivin的定位;随着细胞进入分裂期,GFP信号指示BmSurvivin与染色质共定位,待细胞形成明显的双取向时在纺锤体区域也可以看到GFP信号;后期随着姐妹染色单体分开,GFP信号指示BmSurvivin定位在染色质及胞质分裂区;末期随着两个新的子细胞形成GFP信号指示BmSurvivin仅定位在胞质分裂区。H3Ser10ph定位在BmN4细胞前中期染色质凝缩处,至中期信号最强,与整条染色体重合,后期信号消失。【结论】BmSurvivin与有丝分裂周期中染色质和纺锤体的动态变化相关。     

The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue

The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.     

家蚕细胞中过表达家蚕核型多角体病毒核衣壳蛋白VP39抑制BmNPV的增殖

【目的】家蚕Bombyx mori核型多角体病毒（BmNPV）核衣壳蛋白VP39为病毒装配所必需。本研究旨在初探VP39在病毒侵染家蚕细胞过程中的功能及特征,以期为家蚕抗病毒研究提供研究基础。【方法】本研究通过构建原核表达载体,诱导原核表达得到多克隆抗体,以Western blot验证VP39表达时相;构建真核表达载体,转染细胞后以免疫荧光手段观测VP39表达定位及影响病毒增殖现象。【结果】制备了VP39多克隆抗体。VP39在病毒感染后大量定位于家蚕细胞核,部分定位于胞质,而过表达的VP39定位于家蚕细胞胞质;过表达VP39后抑制BmNPV感染家蚕细胞。【结论】在BmN-SWU1细胞中过表达VP39会影响BmNPV的扩散,导致BmNPV感染细胞数目大量减少。该结果为VP39调控宿主与病毒的相互作用提供了新的思路。     

家蚕核型多角体病毒遍在蛋白及结合肽的研究

从家蚕核型多角体病毒镇江株(Bombyx mori nuclear polyhedrosis virus Zhenjiang strain, BmNPV-ZJ)基因组DNA中克隆遍在蛋白（BmVUB）基因.序列分析结果显示,BmVUB基因长234 bp,编码77个氨基酸.BmVUB氨基酸序列内有一致HTH序列,遍在蛋白保守序列LRLRGG,参与遍在蛋白-蛋白酶复合体形成的4个保守性功能位点(Lys-29、Cys-48、Cys-63、Gly-76)及保守的Gly-Gly-X（X是疏水氨基酸残基）蛋白酶切信号序列,在遍在蛋白保守的Gly-Gly后多出由一个氨基酸组成的延伸肽.原核诱导表达表明BmVUB主要以可溶性形式存在,表达量占总菌蛋白的50%以上.纯化的遍在蛋白浓度为1.03～2.46 g/L,制备抗体,效价在3.2×10^-5以上.用噬菌体表面展示技术筛选遍在蛋白结合肽,所筛选的结合肽可与遍在蛋白特异性结合,其中结合肽VAPHHAYAPMRT对细胞增殖有明显的浓度调控作用,即低浓度促进细胞生长,高浓度强烈抑制细胞的生长.     

Biochemical characterization of rab proteins from Bombyx mori

The small GTPases known as Rab proteins are key regulators of membrane trafficking. We used RT-PCR to isolate cDNA clones of insect-specific Rab proteins (BRabN1 and BRabN2) showing low homology with known Rab proteins from other animals, from mRNA of Bombyx mori. These 2 Rabs were produced in Escherichia coli and purified. BRabN1 bound [(3)H]-GDP and [(35)S]-GTPgammaS with dissociation constants of 0.087 x 10(-6) M and 1.02 x 10(-6) M, respectively, whereas those of BRabN2 were 0.546 x 10(-6) M and 1.02 x 10(-6) M, respectively. Binding of [(35)S]-GTPgammaS to BRabN1 and N2 was inhibited by GDP and GTP. The GTP-hydrolysis activities of BRabN1 and N2 were 154 and 35.5 mmol/min/mole, respectively, and bound [(35)S]-GTPgammaS was exchanged efficiently with GTP. BRabN1 also showed ATPase activity and exchange of [(35)S]-GTPgammaS with ATP. Monoclonal antibodies against BRabN1 and N2 did not recognize any other Rab proteins, and Western blotting using the anti-BRabN1 antibody revealed a single band in the testis of B. mori. These results suggest that BRabN1 and N2 of B. mori bind GTP, convert from the GTP-bound state to the GDP-bound state by intrinsic GTP hydrolysis activity, and return to the GTP-bound state with the exchange, and that BRabN1 is specifically expressed in testis. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.     

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.     

Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain

Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90?% on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.     

家蚕抗BmNPV品系与感性品系血淋巴液蛋白质组的差异分析

利用遗传学的原理, 通过杂交和回交的方法, 建立家蚕抗BmNPV、感BmNPV以及近等基因系模型, 利用2-D电泳和MALDI TOF/TOF MS质谱技术, 从蛋白质组水平上研究家蚕对BmNPV抵抗性。其结果是获得家蚕高抗NB, 高感306, 近等基因系BC8五龄起蚕血淋巴液蛋白质差异表达谱, 分别获得180、190、187个蛋白点, 其中80%的蛋白点集中在等电点5~9范围之内。从三块凝胶上共获得明显差异蛋白点12个, 由质谱鉴定出5种蛋白, 其中氨基酰化酶(Aminoacylase)仅出现在抗性品系NB、近等基因系图谱中, 感性品系没有出现, 初步推测是家蚕抗BmNPV特有蛋白, 这是首次报道结果。     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.     

家蚕抗BmNPV品系与感性品系血淋巴液蛋白质组的差异分析

利用遗传学的原理, 通过杂交和回交的方法, 建立家蚕抗BmNPV、感BmNPV以及近等基因系模型, 利用2-D电泳和MALDI TOF/TOF MS质谱技术, 从蛋白质组水平上研究家蚕对BmNPV抵抗性。其结果是获得家蚕高抗NB, 高感306, 近等基因系BC8五龄起蚕血淋巴液蛋白质差异表达谱, 分别获得180、190、187个蛋白点, 其中80%的蛋白点集中在等电点5~9范围之内。从三块凝胶上共获得明显差异蛋白点12个, 由质谱鉴定出5种蛋白, 其中氨基酰化酶(Aminoacylase)仅出现在抗性品系NB、近等基因系图谱中, 感性品系没有出现, 初步推测是家蚕抗BmNPV特有蛋白, 这是首次报道结果。     

家蚕CVDAR品系对BmNPV的抗性特征分析

【目的】家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)促使的血液型脓病是一种产业上非常严重的家蚕疾病,目前有效的防控方法较少。本研究以大造和CVDAR家蚕品系(对BmNPV有较强抗性的品系)为试验材料,通过分析CVDAR对BmNPV抗性特征,以期确定CVDAR对BmNPV的抗性机制。【方法】本研究通过半致死剂量分析,发现CVDAR品系比大造品系对BmNPV感染的半致死剂量提高10倍以上;进一步HE染色分析大造与CVDAR品系病毒感染前后的中肠组织的变化,具体解析抗性品系CVDAR的抗BmNPV机制。【结果】感染BmNPV 72 h后,大造中肠细胞细胞核明显膨大,着色变浅,到96h后,细胞核持续增大有脱落趋势;而CVDAR抗性品系只在感染96 h后有中肠部分细胞核膨大,但排列整齐;同时通过荧光定量分析大造与CVDAR品系病毒感染后的增殖情况,结合各个时期代表病毒基因的转录水平分析比较发现,感染BmNPV后0–12h也没有发现抗性品系CVDAR和大造之间的病毒拷贝数以及病毒基因转录水平的不同,但感染24h后发现抗性品系CVDAR无论是病毒拷贝数还是病毒基因的转录表达水平都明显低于对照大造。【结论】证明CVDAR口服添毒后中肠中病毒基因的转录在第一轮复制期间不受影响,之后转录水平降低。鉴定CVDAR品系抑制BmNPV增殖的关键时期是在感染BmNPV后的24 h,为解析抗性机制奠定基础。