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.     

粉纹夜蛾颗粒体病毒增效基因3'端2.5 kb片段在大肠杆菌中的表达

将粉纹夜蛾Trichoplusia ni颗粒体病毒增效基因3'端2.5 kb片段插入pQE-31中构建了重组表达载体pQE/enhancin,转化大肠杆菌M15(pREP₄)在IPTG诱导下成功表达出分子量约为96 kD的融合蛋白并命名为P96。初步纯化的P96显示了明显的增效活性,可提高棉铃虫核型多角体病毒对棉铃虫3龄幼虫感染死亡率27.40%～34.50%,缩短LT₅₀ 1.9天以上。     

粘虫颗粒体病毒增效蛋白基因片段优化及功能

【目的】研究转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus,Pu GV-Ps)增效蛋白基因截短片段优化及其增效作用,探索增效蛋白基因的合理利用途径。【方法】生物信息学分析增效蛋白结构域,构建增效蛋白基因截短片段原核表达载体,分析目的基因片段表达产物的表达水平、围食膜蛋白降解效能和增强活性,进一步明确Pu GV-Ps增效蛋白基因的功能区域。【结果】Pu GV-Ps增效蛋白含有M60-like结构域、锌离子催化域和糖蛋白结合域,并包含13个潜在的糖基化位点。以此为依据设计P69(短截M60-like结构域)和P77(短截糖蛋白结合域)2个截短片段,构建了表达载体p ET15b-P69和p ET15bP77,原核表达量明显高于全长基因P104。表达产物纯化蛋白围食膜降解活性表明,P69对斜纹夜蛾围食膜大分子蛋白降解程度高于P77,但两者均低于P104。病毒增强苏云金杆菌(Bt)实验表明,截短片段的表达产物提高了Bt对小菜蛾的毒力,但增强活性显著低于P104。【结论】研究结果表明,Pu GV-Ps增效蛋白基因N端M60-like结构域和C端糖蛋白结合域对其增效作用的发挥都具有一定功能,这些结构对维持增效蛋白的构象也发挥了一定的作用,截短片段P69有利于保持Pu GV-Ps增效蛋白的活性、提高表达水平。该研究结果对增效蛋白的工业化生产具有一定的指导意义。     

粘虫颗粒体病毒增效蛋白在苏云金芽胞杆菌中的表达及增效活性

【目的】在苏云金芽胞杆菌(Bacillus thuringiensis, Bt)中表达截短后的转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus-Ps, PuGV-Ps)增效蛋白,为构建增效Bt工程菌提供理论基础。【方法】通过对截短后增效蛋白的密码子进行优化,构建增效蛋白及其融合蛋白表达载体,分析不同启动子指导下增效蛋白表达量的变化,明确增效蛋白对Bt的增效活性。【结果】本研究构建了表达载体pHTP_cry1AcCoEn81、 pHTRHCoEn81和pHTNCCoEn81, SDS-PAGE结果显示pHTP_cry1AcCoEn81和pHTNCCoEn81分别可以产生81 kDa和134 kDa的重组蛋白。启动子Pcry1Ac和P_cry8E指导下的增效蛋白表达量和重组增效蛋白产量均无显著性差异。生物测定结果表明,重组增效蛋白可以显著增加Bt对小菜蛾的杀虫活性。【结论】研究结果表明,密码子优化的PuGV-Ps增效蛋白可以在Bt中表达并具有显著增效活性,为高效苏云金芽胞杆菌工程菌的构建及...     

棉铃虫颗粒体病毒增效蛋白基因5′端截短片段的表达及增效活性测定

将带有棉铃虫颗粒体病毒增效蛋白 (HaGVenhancin)基因的重组表达载体 pET 30a En分别用BalⅠ和DraⅠ酶切后连接 ,构建重组表达载体pET 30a Ben和 pET 30a Den ,其外源基因的开放阅读框分别为HaGV增效蛋白基因 5′端的 1 7kb和 2 2kb。IPTG诱导表达产生 66 7kD和 85 1kD的多肽并命名为Ben和Den。初步纯化的Ben和Den显示了对棉铃虫核多角体病毒 (HaNPV)及Bt的增效活性。Ben可对棉铃虫核多角体病毒增效 1 0 5%～2 6 5% ,LT50 缩短 0 9d ,Den增效 1 0 2 %～ 33 0 % ,LT50 缩短 1 2d～ 1 9d。重组增效蛋白可对Bt(1∶2 50 0稀释 )增效 2 0 7%～ 35 4% ,Den增效 1 6 7%～ 31 5% ,Ben增效 1 1 7%～2 7 4%。这为进一步研制抗虫工程菌打下了良好的基础     

棉铃虫颗粒体病毒增效蛋白基因5'端截短片段的表达及增效活性测定

将带有棉铃虫颗粒体病毒增效蛋白(HaGV enhancin)基因的重组表达载体pET-30a-En分别用Bal Ⅰ和Dra Ⅰ酶切后连接,构建重组表达载体pET-30a-Ben和pET-30a-Den,其外源基因的开放阅读框分别为HaGV增效蛋白基因5'端的1.7kb和2.2kb.IPTG诱导表达产生66.7kD和85.1kD的多肽并命名为Ben和Den.初步纯化的Ben和Den显示了对棉铃虫核多角体病毒(HaNPV)及Bt的增效活性.Ben可对棉铃虫核多角体病毒增效10.5%～26.5%,LT50缩短0.9d,Den增效10.2%～33.0%,LT50缩短1.2d～1.9d.重组增效蛋白可对Bt(12500稀释)增效20.7%～35.4%,Den增效16.7%～31.5%,Ben增效¨.7%～27.4%.这为进一步研制抗虫工程菌打下了良好的基础.     

棉铃虫颗粒体病毒增效蛋白基因2.6kb片段的表达

以棉铃虫颗粒体病毒(Helicoverpa armigera granulosis virus,简称HaGV)基因组的DNA为模板设计引物,PCR扩增病毒增效蛋白(Enhanicn)基因,然后经BamHI/Pst I双酶切消化,得到近乎全长的约2.6kb的增效蛋白基因片段,再与pQE32质粒连接,构建了重组表达载体pQE2/En,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为102kD的融合蛋白并命名为P102,纯化的P102包涵体显示了明显的增效活性,在感染后168h时统计可提高HaNPV对棉铃虫幼虫的感染死亡率6.25%～27.09%,缩短LT5012.3h以上;在感染后72h时统计可提高Bt对棉铃虫幼虫的感染死亡率28.18%,缩短LT5o12.33h.     

小菜粉蝶颗粒体病毒颗粒体蛋白基因的序列测定及在大肠杆菌中的表达

根据颗粒体病毒颗粒体蛋白(Granulin)基因在其起始密码子上游的12个碱基高度保守序列(TATAAGGAATTT)以及大菜粉蝶颗粒体病毒(PbGV)的颗粒体蛋白基因的序列[1]设计引物,PCR扩增得到850bp左右大小的片段,核苷酸序列测定结果表明该病毒的granulin基因全长为855bp,起始密码位于第38~40位碱基,终止密码位于779~781位碱基,编码框序列全长为744;推测该基因编码一段由247个氨基酸组成的多肽,分子质量约为2.9178×104道尔顿。与其它颗粒体病毒颗粒体蛋白基因进行同源性比较,核苷酸同源性都在70%以上,氨基酸同源性都在75%以上,最高的为大菜粉蝶颗粒体病毒(PbGV),核苷酸同源性为97%,氨基酸同源性为98%。构建了重组表达载体pet-28a-Gran,IPTG诱导后经SDS-PAGE检测,表明获得了颗粒体蛋白基因在大肠杆菌BL21中的特异表达。     

棉铃虫颗粒体病毒增效蛋白基因2.6kb片段的表达

以棉铃虫颗粒体病毒（Helicoverpa armigera granulosis virus,简称HaGV）基因组的DNA为模板设计引物,PCR扩增病毒增效蛋白（Enhanicn）基因,然后经BamH Ⅰ/Pst Ⅰ双酶切消化,得到近乎全长的约2.6kb的增效蛋白基因片段,再与pQE32质粒连接,构建了重组表达载体pQE32/En,转化大肠杆菌M15(pREP4),在IPTG诱导下表达出分子量约为102kD的融合蛋白并命名为P102,纯化的P102包涵体显示了明显的增效活性,在感染后168h时统计可提高HaNPV对棉铃虫幼虫的感染死亡率6.25%-27.09%,缩短LT5012.3h以上;在感染后72h时统计可提高Bt对棉铃虫幼虫的感染死亡率28.18%,缩短LT5012.33h。     

Transgenic tobacco transformed with the Trichopusia ni Granulovirus Enhancin gene affects insect development

Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demonstrated to enhance baculovirus infection in larvae. In this paper we describe the effect of the long-term feeding of lyophilized transgenic tobacco material to Pseudaletia separata and Spodoptera exigua larvae. Our results demonstrated that the baculovirus enhancin gene products have potential for use in insect pest management.     

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.     

A new nodavirus-negative Trichoplusia ni cell line for baculovirus-mediated protein production

Cell lines derived from Trichoplusia ni (Tn) are widely used as hosts in the baculovirus-insect cell system (BICS). One advantage of Tn cell lines is they can produce recombinant proteins at higher levels than cell lines derived from other insects. However, Tn cell lines are persistently infected with an alphanodavirus, Tn5 cell-line virus (TnCLV), which reduces their utility as a host for the BICS. Several groups have isolated TnCLV-negative Tn cell lines, but none were thoroughly characterized and shown to be free of other adventitious viruses. Thus, we isolated and extensively characterized a new TnCLV-negative line, Tn-nodavirus-negative (Tn-NVN). Tn-NVN cells have no detectable TnCLV, no other previously identified viral contaminants of lepidopteran insect cell lines, and no sequences associated with any replicating virus or other viral adventitious agents. Tn-NVN cells tested negative for >60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma. Finally, Tn-NVN cells grow well as a single-cell suspension culture in serum-free medium, produce recombinant proteins at levels similar to High Five™ cells, and do not produce recombinant glycoproteins with immunogenic core α1,3-fucosylation. Thus, Tn-NVN is a new, well-characterized TnCLV-negative cell line with several other features enhancing its utility as a host for the BICS.     

Enhancement of Bacillus thuringiensis Toxicity to Lepidopterous Species with the Enhancin from Trichoplusia ni Granulovirus

The enhancin from the Trichoplusia ni (Hübner) granulovirus (TnGV) is a 104-kDa viral protein that can significantly increase the virulence of several baculoviruses. Enhancin can destroy the structural integrity of the peritrophic membrane (PM) of lepidopterous larvae, resulting in more efficient penetration of the PM by virions and avoidance of inactivation by larval midgut factors. In this study we demonstrate that TnGV enhancin can also enhance toxicity of Bacillus thuringiensis (Berliner) (Bt) to several noctuid species. Addition of enhancin significantly increased the toxicity of Dipel, a commercial Bt formulation, to six species (T. ni, Helicoverpa zea (Boddie), Heliothis virescens (Fabricius), Spodoptera exigua (Hübner), Pseudoplusia includens (Walker), and Anticarsia gemmatalis (Hübner)). Furthermore, the effectiveness of four commercial Bt-based products (Cutlass, Javelin, Biobit, and Dipel) against T. ni was enhanced three- to sixfold by adding purified enhancin. We hypothesize that the PM of larvae can impede the movement of the BT toxin protein to the midgut brush border and that enhancin can increase the toxicity by affecting the permeability of the PM.     

Proteomics analysis of Trichoplusia ni midgut epithelial cell brush border membrane vesicles

The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinfbrmatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel elec? trophoresis coupled with multi-dimensional protein identification technology. More than 3000 proteins were associated with the BBMV of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI-anchor signal. Of these, bioinfbrmatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell-cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.     

粉纹夜蛾离体细胞抗菌肽的抗菌谱测定

用热灭活的大肠杆菌DHSQ诱导粉纹夜蛾(Trichoplusia ni)离体细胞产生抗菌肽,用三氯乙酸沉淀法提取出该活性物质,采用琼脂糖孔穴扩散法和生长抑制测定法测定其抗菌谱,发现该抗菌物质具有较广的抗微生物活性,其中特别是对革兰氏阴性菌中的沙门氏茵和大肠杆茵,酵母菌中的白色念珠菌,植物病源真茵中的花生白绢病茵和小麦赤霉病茵具有较强的抑菌活性,从而表明该物质是一种既抗细菌,又抗真菌的抗微生物肽。     

异源病毒感染对银纹夜蛾血淋巴酯酶的影响

对染病昆虫酯酶同工酶（简称酯酶）的变化进行分析测定,已成为了解病毒进入虫体靶器官后的病理生化变化以及病毒复制与虫体新陈代谢之间关系的重要途径之一,这方面的报道“J侧重于同源病毒——寄主系统的研究,本研究则针对银纹夜蛾（AWrammaagnata）幼虫感染异源粉纹夜蛾核型多角体病毒（TnNPV）后血淋巴酯酶的变化进行了探讨。现将结果报告如下：材料和方法1材料11虫源实验室内用半人工饲料饲养三代的健康银纹夜蛾五龄村幼虫。l．2毒源由中山大学昆虫研究所生物工程室提供的已纯化的TnNPV病毒株。1．3幼虫血淋巴样品的制备挑选工龄…     

Plant exudates trigger leaf-trenching by cabbage loopers, Trichoplusia ni (Noctuidae)

Cabbage loopers, Trichoplusia ni, cut a narrow trench across leaves of plants that release exudate, then feed distal to the trench in an area of reduced exudation. The larvae do not normally trench plant species such as plantain, Plantago lanceolata, that lack exudate. To determine what cues elicit trenching, I reared larvae to the final instar on plantain, then applied test solutions to their mouthparts during feeding. Loopers that received latex from Lactuca serriola (Asteraceae) or phloem exudate from watermelon, Citrullus vulgaris (Cucurbitaceae), often responded by cutting a trench in plantain, even though these larvae had not previously encountered exudate nor previously trenched. Loopers that were allowed to trench and feed on L. serriola for 1 day prior to the assay subsequently cut trenches in plantain more frequently and in response to more fluids, including a viscous solution of polyethylene glycol and latex from a non-host, poinsettia (Euphorbia pulcherrima). Subsequent bioassays with larvae reared entirely on plantain tested whether bitter cucurbitacins or gelation are essential cues for trenching. Sap from non-bitter cucumber plants (Cucumis sativus) caused larvae to trench, showing that cucurbitacins are not required to induce trenching. Loopers also trenched after receiving cucumber sap that did not gel due to the addition of mercaptoethanol. An extract of sap lacking the proteins that cause gelation likewise triggered trenching. Further fractionation revealed that cucumber sap and also butternut squash sap (Cucurbita moschata) contain trenching stimulants that are small (molecular weight < 3,000) water-soluble molecules. Received: 13 January 1997 / Accepted: 6 June 1997