人促红细胞生成素基因在家蚕体中的高效表达

人促红细胞生成素(EPO)是一种调控红系干细胞增殖、分化和成熟的糖蛋白激素。将合成的EPO cDNA插入杆状病毒转移载体pBlueBacⅢ,使其置于Ph基因强启动子控制之下,获得了转移载体pBlueBacEPO。将pBlueBacEPO DNA与野生型BmNPV DNA共转染BmN细胞,经空斑纯化,获插入EPO cDNA的重组病毒rBmNPVEPO。经Sonthern杂交和PCR扩增鉴定证明人EPO基因已正确组建于BmNPV的预定位置。将重组病毒rBmNPVEPO穿刺接种5龄幼虫和蛹,收集感染第3～5d的幼虫血淋巴和3～6.5d蛹血淋巴。用ELISA检测幼虫血淋巴中EPO表达量高达62800u/mL,蛹血淋巴中表达量达74000u/mL。Western blot结果显示幼虫血淋巴和蛹血淋巴均有一条明显的免疫杂交带,分子量均约为26kD。用TF1细胞对幼虫表达产物进行了生物活性测定,每毫升血淋巴中EPO活性约为63000u。     

The effect of nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes on hemolymph proteins, sugars, and lipids in Spodoptera exigua larvae

We determined the physiological effects of joint and separate nucleopolyhedrovirus (NPV) infection and parasitism by the endoparasitoid Microplitis pallidipes Szepligeti on biochemical events in the noctuid Spodoptera exigua (Hübner). The results indicated that in parasitized larvae, compared to healthy larvae, total protein concentration in host hemolymph began to decline and total sugar concentration significantly increased by the first day, while lipid content in the host body significantly increased by the second day after parasitization. Meanwhile, in jointly infected and parasitized hosts, compared to parasitized larvae, total protein concentration was consistently higher, total sugar concentration was consistently lower, and lipid content became higher by the second day after treatment. In virus-infected larvae, compared to healthy larvae, total protein concentration sharply declined during the first two days but increased by the third, while total sugar concentration increased on the second and third days after virus infection but decreased at other observation times, and lipid content began to increase by the second day after virus infection. Finally, in larvae that were both parasitized and virus-infected, compared to just virus-infected larvae, total protein concentration increased during the first two days but decreased by the third, total sugar concentration increased only on the first and fourth days, and lipid content decreased significantly on the first day but began to increase by the second day after treatment. These findings led us to conclude that parasitization inhibited protein mobilization but stimulated sugar mobilization in host hemolymph, and promoted lipid mobilization in the host body, while Spodoptera exigua NPV infection stimulated protein mobilization induced by parasitization but inhibited sugar mobilization induced by parasitization.     

Isolation and characterization of bacteria-induced protein P4 from hemolymph of Manduca sexta

Insects synthesize several types of hemolymph proteins in response to bacterial infection. The objective of this study was to characterize a 48,000 dalton hemolymph protein induced in larvae of Manduca sexta after injection of bacteria. The protein, isolated by cation exchange and gel filtration chromatography from hemolymph of larvae injected with Micrococcus lysodeikticus, was found to be a glycoprotein with pI = 8.4. The molecular weight, isoelectric point, amino acid composition, and NH2 terminal sequence of the protein are similar to bacteria-induced protein P4 from Hyalophora cecropia, and the M. sexta protein is also designated P4. The hemolymph concentration of M. sexta P4 (35 +/- 7 micrograms/ml in day 3 fifth instar larvae) increases 30- to 45-fold by 48 h after injection of bacteria, but it does not increase in response to injection of distilled water. Lower levels of induction occur after injection of peptidoglycan fragments, zymosan, and lipopolysaccharide. The properties of M. sexta P4 are very similar to those of a previously characterized M. sexta hemolymph protein known as postlarval protein, and antibodies against P4 bind to post-larval protein.     

Concomitant primary infection of the midgut epithelial cells and the hemocytes of Trichoplusia ni by Autographa californica nucleopolyhedrovirus

We have constructed a modified Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to express the green fluorescent protein (GFP) under the polyhedrin promoter and used it to study the infection process of AcMNPV in Trichoplusia ni larvae. T. ni larvae that ingested the virus showed localized expression of GFP in the midgut epithelial cells and the hemocytes at 12 h post infection (hpi). The presence of GFP-related fluorescence in the midgut columnar cells indicated that the virus was not only replicating, but also synthesizing the late viral proteins. Studies using the transmission electron microscope showed that the virus infected the midgut columnar cells. At the same time a proportion of the parental virus travelled through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, entered the hemocoel and infected the hemocytes. This resulted in the simultaneous infection of the midgut epithelial cells and the hemocytes. Subsequently, the budded virus (BV) released from the infected hemocytes into the hemolymph caused secondary infection within the tracheal epithelial cells. The virus then rapidly spread through the tracheal system allowing the infection of a variety of other tissues such as the epidermis and the fat body.     

Isolation and characterization of constituents in hemolymph from healthy and diseased japanese beetle larvae

Studies are reviewed on the composition of hemolymph from Japanese beetle larvae, Popillia japonica. This research was conducted to determine the chemical environment required for sporulation of the milky disease organism, Bacillus popilliae. Amino acids, proteins, organic acids, lipids, carbohydrates, and oxygen content were determined in hemolymph from healthy and diseased larvae. Oxygen content of hemolymph was measured by microelectrodes implanted in the hemocoel of living larvae. Vegetative growth of the pathogen reduces the dissolved oxygen; however, as sporulation occurs, oxygen concentration approaches normal values. Trehalose, the major hemolymph sugar, is reduced by ca. 50% during the course of milky disease. Neutral lipids, phospholipids, and hydrocarbons decrease markedly with infection. Both branched-chain and normal alkanes occur in hemolymph. Milky disease causes an increase in concentration of pyruvic, malic, glycolic, tartaric, and glyoxylic acids. The major hemolymph protein, a lipoglycoprotein, has a mol. wt. of ca. 500,000. Reduced concentration of this protein is accompanied by the appearance of a smaller protein. Amino acids that increase significantly as a result of infection are: Glutamic acid, aspartic acid, β-alanine, phenylalanine, threonine, lysine, and serine. Hemolymph contains few peptides of low molecular weight.     

淡足侧沟茧蜂寄生对甜菜夜蛾幼虫血淋巴总糖、蛋白质及虫体脂质含量的影响

为探讨淡足侧沟茧蜂Microplitis pallidipes Szepligeti调控寄主的生理机制,测定了淡足侧沟茧蜂寄生后,甜菜夜蛾Spodoptera exigua(Hübner)幼虫血淋巴总糖、蛋白质及虫体脂类含量的变化。研究结果表明,寄生后连续5 d的观察时间内,甜菜夜蛾幼虫血淋巴总糖含量从寄生后第1天开始就高于未寄生寄主幼虫,且在寄生后第2至5天达到显著水平;除寄生后第3天外,被寄生寄主幼虫血淋巴总蛋白质含量始终低于未寄生幼虫,且在寄生后第1、4、5天达到显著水平;甜菜夜蛾被寄生后,虫体脂质含量始终高于未寄生幼虫,且从寄生后第2天开始达到显著水平。本研究揭示,淡足侧沟茧蜂寄生刺激了寄主甜菜夜蛾幼虫血淋巴总糖合成,但抑制了其蛋白质合成,同时也刺激了甜菜夜蛾虫体脂质合成。     

烟青虫核型多角体病毒的复制和染病后血淋巴蛋白的变化

用烟青虫(Hiliothis assulta)核型多角体病毒感染5龄初烟青虫幼虫,以染病后24、48·,72、96/120小时分别测定了虫体血淋巴蛋白浓度的变化。幼虫染病后24小时血淋巴蛋白浓度要高于同期对照组,72小时后染病幼虫血淋巴蛋白浓度较对照急剧下降。在染病及对照血淋巴样品中电泳分析(PAGE)三种蛋白(普通蛋白、糖蛋白和脂蛋白)均可分别染出22条、3条和3条带。分析结果表明,在虫体正常生长代谢过程中发生变化的主要蛋白可能大多为糖脂复合蛋白,但病毒的侵染能抑制这些变化的产生。电镜观察染病毒后幼虫的中肠组织和气管上皮组织,发现中肠染病轻微,不形成多角体。气管上皮细胞感染情况表明其属于对NPV感染较为敏感的组织之一,并在虫体染病后的病毒二次感染上可能起着重要作用。     

Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae

It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVL-infected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ≤ vERTVL < vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae.     

Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp.

Monoclonal antibodies (MAbs) were produced against white spot syndrome virus (WSSV) of penaeid shrimp. The virus isolate used for immunization was obtained from China in 1994 and was passaged in Penaeus vannamei. The 4 hybridomas selected for characterization all produced MAbs that reacted with the 28 kD structural protein by Western blot analysis. The MAbs tested in dot-immunoblot assays were capable of detecting the virus in hemolymph samples collected from moribund shrimp during an experimentally induced WSSV infection. Two of the MAbs were chosen for development of serological detection methods for WSSV. The 2 MAbs detected WSSV infections in fresh tissue impression smears using a fluorescent antibody for final detection. A rapid immunohistochemical method using the MAbs on Davidson's fixed tissue sections identified WSSV-infected cells and tissues in a pattern similar to that seen with digoxigenin-labeled WSSV-specific gene probes. A whole mount assay of pieces of fixed tissue without paraffin embedding and sectioning was also successfully used for detecting the virus. None of the MAbs reacted with hemolymph from specific pathogen-free shrimp or from shrimp infected with infectious hypodermal and hematopoietic necrosis virus, yellow head virus or Taura syndrome virus. In Western blot analysis, the 2 MAbs did not detect any serological differences among WSSV isolates from China, Thailand, India, Texas, South Carolina or Panama. Additionally, the MAbs did not detect a serological difference between WSSV isolated from penaeid shrimp and WSSV isolated from freshwater crayfish.     

Expression of post-translational processing of preprocecropin A using a baculovirus vector.

A cDNA fragment encoding preprocecropin A was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in recombinant-infected last instar larvae of Trichoplusia ni and in diapausing pupae of Hyalophora cecropia. The identity of the recombinant product was established by electrophoresis with detection of antibacterial activity and mass spectrometry. The prepropeptide had been correctly processed including removal of signal peptide and pro-part. Biologically active and amidated cecropin A was exported to the hemolymph. The yield of recombinant protein in H. cecropia reached a level of 600 micrograms/ml hemolymph and about 70% of the material was amidated.     

Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an efficient reporter enzyme in vivo

Trichoplusia ni and Spodoptera littoralis larvae were infected with a recombinant AcNPV, having the viral polyhedrin gene replaced with the cDNA encoding firefly luciferase. Both S. littoralis and T. ni synthesized very high levels of luciferase representing greater than or equal to 25% and greater than or equal to 15%, respectively of the total Coomassie blue stainable protein. Luciferase was apparently not secreted into the hemolymph but was contained within the body tissue. Expression in S. littoralis larvae suggests that luciferase can be an excellent reporter enzyme to study virus infection, dissemination and expression in different tissues, host range determination, insect physiology and also to monitor the release of recombinant virus in the environment when used as a biocide.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

保幼激素类似物对斜纹夜蛾核型多角体病毒在宿主血淋巴中增殖动态的影响

通过研究保幼激素类似物(juvenile hormone analogues, JHA) methoprene对斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）在宿主血淋巴中增殖的影响,以探明JHA促进 SpltNPV增殖的初步机制,为阐明JHA促进病毒增殖提供更全面的理论依据。应用SDS PAGE及Western blot法,分析了methoprene对SpltNPV多角体蛋白(polyhedron, POLH)在宿主斜纹夜蛾6龄幼虫血淋巴中合成的影响。结果表明：经methoprene处理后2～3天可明显促进幼虫血淋巴液中POLH的合成。在此基础上,通过荧光定量PCR检测,发现methoprene对SpltNPV在幼虫血淋巴液复制的影响主要发生在处理后的第4和第5天,该期间polh基因的拷贝数比对照显著增加,拷贝数的峰值达1.22×10¹⁰/mL。     

粘虫幼虫血淋巴中的凝集素

粘虫Mythlmna separata Walker幼虫血淋巴中含有凝集某些脊椎动物红细胞的凝集素,凝集活性可被乳糖、岩藻糖或神经氨酸抑制.用CNBr-sepharose 4B 进行亲和层析从血淋巴中分离的凝集素成分比较复杂,聚丙烯酰胺凝胶电泳显示三条区带,SDS聚丙烯酰胺凝胶电泳出现6个亚基,亚基分子量分别为71000、65000、56000、35000、33000及31000道尔顿.     

饥饿胁迫下家蚕5龄起蚕中的蛋白表达谱及 BmLp-c 6的转录分析

【目的】分析家蚕Bombyx mori受饥饿胁迫后的蛋白质谱变化,探索其耐受饥饿的机理。【方法】以家蚕品种932为实验材料,利用双向电泳和质谱技术检测5龄起蚕经过24 h饥饿胁迫的蛋白质谱差异变化,利用荧光定量PCR技术分析BmLp-c 6的转录表达。【结果】经比对饥饿蚕和正常取食蚕的血淋巴蛋白谱,饥饿蚕有62个特异蛋白点。蛋白点的等电点在4.22~6.98之间,分子量分布在20.81～144.69 kDa间。选取只在饥饿时出现的特异蛋白点No. 7111进行质谱鉴定,根据其氨基酸序列进行引物设计,获得了目的基因BmLp-c 6,经与载体pET-21d(+)连接重组后,成功获得诱导表达。经实时荧光定量PCR分析,当5龄起蚕受到饥饿胁迫影响时,BmLp-c 6基因在血淋巴中大量转录表达,但在中肠中的转录表达水平却极低。【结论】家蚕5龄起蚕在饥饿胁迫下,血淋巴中的蛋白质谱发生变化,BmLp-c 6会大量转录表达。     

Changes in <Emphasis Type="Italic">Galleria mellonella</Emphasis> lysozyme level and activity during <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> infection

The level of lysozyme in fat body, hemocytes and cell-free hemolymph from Galleria mellonella larvae infected with Pseudomonas aeruginosa was determined and evaluated. In the samples of fat body and hemocytes, an increase in lysozyme content was detected 1 d after infection and then a significant decrease was observed after a prolonged infection time. In the case of cell-free hemolymph, an increase in the lysozyme level was noticeable during the first 30 h post injection and stayed at a similar level for 42 h. The smaller decrease of the lysozyme level after 42 h might be associated with the development of bacteremia of P. aeruginosa in insects. In addition, the gradual increase in the content of lysozyme correlated with the increase of its activity in the hemolymph of the infected larvae as a response to injection with P. aeruginosa. The G. mellonella lysozyme appeared to be insensitive to extracellular proteinases produced in vivo by P. aeruginosa.     

利用杆状病毒表达系统表达金鱼生长激素I基因

以不含起始密码ATG的质粒Psxivvi⁺X3为转移载体,将编码金鱼生长激素I的Cdna插入粉纹夜蛾核型多角体病毒(TnNPV)基因组中,构建了形成多角体的重组病毒株TnNPV—SX⁺gfGHl21a。该毒株能利用合成多角体XIV串联启动子,在重组病毒感染的草地贪夜蛾(Spodoptera frugiperda,Sf)昆虫细胞及银纹夜蛾幼虫中表达金鱼生长激素I基因。感染离体细胞及虫俸后的蛋白SDS聚丙烯酰胺凝胶电泳表明．所表达的蛋白分子量为22．5kDa,与理论计算值相符。Westem印迹证实。金鱼生长激素特异蛋白得到表达。     

Production of Classical Swine Fever Virus Envelope Glycoprotein E2 as Recombinant Polyhedra in Baculovirus-Infected Silkworm Larvae

Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68?mg/ml of hemolymph and 0.53?mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.     

RNA interference as a metabolic engineering tool: potential for in vivo control of protein expression in an insect larval model

Many ex vivo factors influence the yield of recombinant protein produced via AcMNPV (Autographa californica multiple nucleocapsid nuclear polyhedrosis virus) in Trichoplusia ni (T. ni) larvae. Among these are: the method of infection, the time of infection, the virus load, and the time of harvest. In vivo strategies, however, that attempt to manipulate host function in this and other expression systems have largely been ignored. In this work, RNA interference (RNAi) is shown as an effective metabolic engineering controller to downregulate targeted gene expression. Specifically, RNAi was made to virus-encoded gfp(uv) and was found to inhibit the production of GFPuv in larvae when injected within an 18-h window (before and after) of baculovirus infection. The level of inhibition was found to depend, both in duration and extent, on the concentration of injected RNAi. That relatively low levels of RNAi can inhibit protein synthesis driven by the strong polyhedrin (polh) promoter of AcMNPV, suggests that RNAi will find utility as an in vivo metabolic controller in metabolic engineering studies such as this one pertaining to protein expression.