昆虫颗粒体病毒增效蛋白研究进展

昆虫颗粒体病毒的颗粒体中有一种可以提高核型多角体病毒侵染能力的蛋白质,叫做增效蛋白.后来的研究发现,增效蛋白也可以提高苏云金杆菌等生物杀虫剂的杀虫活性.本文就增效蛋白的性质、基因结构和表达、增效机理,以及增效蛋白对核型多角体病毒和Bt的增效作用等方面的研究进展进行了概述.最后本文还讨论了增效蛋白可能的开发和应用前景.     

Transgenic tobacco plants carrying a baculovirus enhancin gene slow the development and increase the mortality of Trichoplusia ni larvae

This study was done to assess insect growth and mortality on tobacco plants transformed with baculovirus enhancin genes, as a first step toward the possible use of enhancin transgenes as part of an insect control system. Enhancin genes from Trichoplusia ni or Helicoverpa armigera baculoviruses were introduced into tobacco via Agrobacterium tumefaciens with kanamycin selection. PCR analyses of genomic DNA confirmed the presence of the enhancin genes in the kanamycin-resistant plants; however, the expression of the genes was very low and could be detected only with RT-PCR. Bioassays with Trichoplusia ni larvae showed that larval growth and development was significantly slower on some transgenic lines and that larval mortality was higher. The majority of the enhancin-transgenic plants had little or no inhibitory effect. The low expression of enhancin in plants carrying current expression cassettes and the relevance of these results to pest management are discussed.     

Transgenic expression of Dendrolimus kikuchii nucleopolyhedrovirus enhancin in tobacco enhances the mortality and affects the development of Spodoptera exigua larvae

A novel 2551-bp enhancin gene designated En-Dk was isolated from the Dendrolimus kikuchii nucleopolyhedrovirus genome. Sequence analysis revealed that En-Dk encodes a polypeptide chain of 792 amino acid residues containing a conserved enhancin domain and an α-helical transmembrane domain, but no signal peptide. Compared with wild-type tobacco (Nicotiana tabacum L.), transgenic tobacco showed significant phenotypic changes following En-Dk overexpression with slower growth and delayed initial flowering. An interesting finding is that the transgenic tobacco exerted a toxic effect on newly hatched Spodoptera exigua larvae, with a significant inhibitory effect on third-instar larvae in terms of body weight, pupal weight, pupation and the emergence process.     

TnGV增强蛋白在AcMNPV中的表达和活性分析

为了研究杆状病毒增强蛋白的活性基团 ,本文构建了分别表达三种N端部分缺失的粉纹夜蛾颗粒体病毒增强蛋白的重组杆状病毒 ,这三种蛋白在N端分别缺失了 15 0 ,186和 2 5 0个氨基酸。用重组病毒感染Tn 5B1 4细胞 ,成功地表达了这三种蛋白 ,并得到了纯化的蛋白质。通过体外降解围食膜的方法检测这些部分缺失的增强蛋白的活性 ,结果证实这三种蛋白均失去了增强蛋白的降解围食膜粘蛋白的活性。这一结果表明 ,增强蛋白的N端对其降解围食膜粘蛋白的功能是必需的     

Plant-mediated alteration of the peritrophic matrix and baculovirus infection in lepidopteran larvae

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18 h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.     

Uptake of juvenile hormone esterase by pericardial cells of Manduca sexta

Immunohistochemical studies were conducted to determine tissue(s) which might be involved in the uptake of juvenile hormone esterase (JHE) from larval hemolymph. Purified JHE expressed by a recombinant baculovirus carrying the JHE gene from Heliothis virescens was injected into the hemolymph of second stadium larvae of Manduca sexta. Immunoreactive material detected with specific antibodies against the natural JHE purified by affinity chromatography from the hemolymph of H. virescens was localized only in the dorsal regions of whole larval mounts. Further immunohistochemical studies of whole and dissected larvae at the light and electron microscopic level showed the specific localization of JHE in pericardial cells. Western blot analysis confirmed the localization of injected JHE in pericardial cells and also indicated some apparent degradation of the incorporated JHE. Similar results were obtained with the JHE from H. virescens injected into larvae of H. virescens. These results indicate that pericardial cells are involved in the uptake of injected JHE from insect hemolymph and its degradation.     

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.     

Effect of a nuclepolyhedrovirus of Autographa californica expressing the enhancin gene of Trichoplusia ni granulovirus on T. ni larvae

A recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) strain showing higher virulence against Trichoplusia ni larvae than the wild-type virus was developed. The 'enhancin' (VEF) gene of T. ni granulovirus (TnGV) and the AcMNPV polyhedrin gene were cloned into the baculovirus transfer vector pAcUW31. This plasmid and AcMNPV BacPAK6 DNA were co-transfected into the BTI-Tn5B1-4 cell line. A recombinant AcMNPV strain (BacVEFPol) was purified, amplified, and bioassayed against T. ni first instar larvae. Its estimated LC₅₀ (0.184 OB/mm²) was 2.18 times lower than the LC₅₀ estimated for the wild-type AcMNPV (0.402 OB/mm²). Likewise, an LT₅₀ of 67.7 h was estimated for the recombinant AcMNPV strain while the LT₅₀ of wild-type AcMNPV was estimated at 81.9 h. This indicates a 17.4% reduction of the time required to kill the larvae. The higher virulence of the recombinant strain, evidenced by its LC₅₀ and LT₅₀ values being lower than those of the wild-type strain, indicates that the VEF protein is expressed properly and may be occluded in the OBs.     

人白细胞介素-11基因在家蚕培养细胞和虫体内的表达

将缺少编码信号肽序列的人白细胞介素－11（hIL-11)546核苷酸cDNA,重组于质粒pBacPAK8构建重组转移载体pBacIL-11,与经线性化修饰的家蚕核型多角体病毒（BmBacPAK)DNA共转染家蚕培养细胞株BmN,获得了插入hIL-11基因的重组病毒。Southern杂交表明重组病毒基因组中含有hIL-11基因片段,RNA斑点杂交表明hIL-11基因得到了转录。重组病毒感BmN细胞株、家蚕幼虫和蛹,在细胞培养上清、细胞抽提物、幼虫和蛹的体液样品中,SDS－PAGE电泳分析都能检测得到表达产物的特异性条带;采用IL－11依赖细胞株B9－11和MTT法测定表达产物的生物活性,表明rIL-11基因分别在培养细胞和蚕体内得到了高效表达。     

A baculovirus enhancin alters the permeability of a mucosal midgut peritrophic matrix from lepidopteran larvae

The peritrophic matrix (PM) in lepidopterous larvae may function as a defensive barrier against ingested viral pathogens. PMs isolated from Trichoplusia ni and Pseudaletia unipuncta larvae, were treated with a baculovirus-encoded metalloprotease (enhancin) from Trichoplusia ni granulosis virus (TnGV) and their in vitro permeability to blue dextran and fluorescent-labelled Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined using a dual chamber permeability apparatus. Incubation of T. ni PMs with 0.0, 0.5, 1.0, and 2.0mg/ml enhancin resulted in a blue dextran 2000 flux of 4.4, 6.3, 9.9, and 15.6&mgr;g/mm(2)/h, respectively. In addition, T. ni PMs treated with enhancin were found to be significantly more permeable to fluorescent-labelled AcMNPV than non-treated control PMs. The permeability of T. ni PMs treated with 3.0mg/ml enhancin was 0.017 cumulative percent crossing/mm(2)/h, whereas the permeability of the control PM was below the detectable limit. Similarly, enhancin treatment greatly increased the permeability of P. unipuncta PMs to AcMNPV. These results provide evidence that the PM from two lepidopteran species can block the passage of baculovirions across this matrix thus reducing the probability of larval infection. Furthermore, these results support the hypothesis that enhancin facilitates NPV infection of larvae by altering the permeability of the PM.     

粉纹夜蛾颗粒体病毒增强蛋白锌离子结合域定点突变

粉纹夜蛾颗粒体病毒（Trichoplusia ni granulovirus, TnGV）增强蛋白（enhancin）具有增强病毒感染力的作用。该蛋白包含一个多种杆状病毒增强蛋白都具有的保守结构域HELGH,是典型的金属蛋白酶锌离子结合域,但该结构域对增强蛋白生物活性的重要性尚未得到研究。本研究通过定点突变构建了该结构域的5个氨基酸分别突变为2种不同氨基酸的共10种增强蛋白突变体基因,并用杆状病毒载体进行了重组表达。活性测定发现,10种突变型增强蛋白大部分都丧失了野生型增强蛋白所具有的降解粉纹夜蛾幼虫围食膜粘蛋白的生物学功能,只有1种（第4位G突变为A）保留该生物学活性。这一结果表明锌离子结合域对增强蛋白生物活性具有重要作用,也提示增强蛋白确是一种金属蛋白酶。     

30K蛋白对家蚕细胞及幼虫存活的作用

利用杆状病毒表达系统在家蚕BombyxmoriL.细胞BmN中表达家蚕30K蛋白,以亲本病毒BmPAK6为对照,将重组病毒Bm/r-30K分别感染BmN细胞及家蚕幼虫,观察其感染后不同时间的凋亡及存活率。与对照相比,重组病毒感染后的BmN细胞的存活率明显高于对照组,并且感染的家蚕幼虫存活时间也较长,表明体内过量表达家蚕30K蛋白有助于延长其细胞及幼虫的存活。     

植酸酶基因在家蚕—杆状病毒表达系统中的表达及其酶学特性

将从黑曲霉菌株Aspergillusniger 96 3克隆并经改造后的植酸酶基因在昆虫 -杆状病毒表达系统中表达 ,SDS PAGE电泳检测蚕体和蛹的表达量分别达到 1.4 3g L血淋巴液和 1.90g L血淋巴液。酶活性测定结果表明 ,在蚕体和蛹的表达活性分别为 4 .6 7× 10 8u L血淋巴液和 5 .99× 10 8u L血淋巴液。该酶活性的最适温度范围为 5 0～6 0℃ ,最适pH值为 5 .5～ 5 .0和 2 .5。研究表明杆状病毒系统表达的植酸酶具有耐酸性和抗高温的特性 ,可以用于生产饲用植酸酶。     

小黑杨PsnHB22基因在烟草中的遗传转化研究

HD-Zip转录因子在光信号转导、非生物胁迫、叶片发育等方面发挥重要的作用，HB22转录因子是HD-ZipⅠ亚家族的成员之一。为研究PsnHB22基因的功能，从小黑杨（Populus simonii×P.nigra）cDNA中克隆PsnHB22基因并构建植物表达载体进行烟草（Nicotiana tabacum）的遗传转化，以获得该基因过量表达的转基因株系。对转基因株系进行PCR、qRT-PCR分子检测后观察表型，结果显示在营养生长时期，转基因烟草叶片窄小并且株高显著低于野生型对照。测定转基因烟草及野生型叶片的叶绿素含量，发现转基因烟草叶绿素含量显著高于野生型。由此推测PsnHB22基因在植株高生长、光合作用及叶片的形态建成等过程中起着重要的作用。     

杆状病毒的垂直传播及绿色荧光蛋白在棉铃虫幼虫中的表达

用转座穿梭系统构建了携带绿色荧光蛋白基因（gfp）的重组棉铃虫核型多角体病毒rHa-FGP,以其多角体添食感染棉铃虫3龄幼虫,室内饲养3代,各代均可见自然光下发绿色荧光的棉铃虫幼虫,其中子代不再重复感染。F₀、F₁、F₂代发绿色荧光的棉铃虫幼虫所占百分比分别为34%、20%、8%。提取虫体内的病毒多角体DNA,以PCR和斑点杂交鉴定表明,gfp不仅在亲代棉铃虫体内正常表达,而且在子代幼虫中表达,HaNPV通过卵实现了垂直传播。     

棉铃虫颗粒体病毒增效蛋白基因2.1kb片段在大肠杆菌中的表达

以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组DNA为模板,设计引物PCR扩增病毒增效蛋白(Enhancin)基因,然后经SacI/PstI双酶切消化,得到5′端截短的约2.1kb增效蛋白基因片段,再与pQE30质粒连接,构建了重组表达载体pQE/EnC,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为78×10³ D的融合蛋白并命名为P78,纯化的P78包涵体显示了明显的增效活性,可提高AcMNPV对小菜蛾幼虫的感染率27.88%～32.92%。     

Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus

Abstract:  The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6× His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8  m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses.