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(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.     

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。     

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。     

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.     

表达乙型肝炎病毒表面抗原基因又形成多角体的杆状病毒

用形成包含体(OCC~+)并能利用人工合成启动序列和多角体XIV启动子表达外源基因的转移载体质粒pSXIVVI~+X3,将乙型肝炎病毒表面抗原(HBsAg)基因和多角体基因同时插入无包含体的粉纹夜蛾核型多角体病毒TnNPV-SVI-G基因组中,得到表达HBsAg基因又形成包含体(多角体)的重组毒侏TnNPV-HBs85-OCC~+。与利用野生型多角体启动子表达HBsAg基因的无包含体毒株TnNPV-HBsD4不同,TnNPV-HBs85-OCC~+由于具包含体,能以口服方式大规模感染粉纹夜蛾(Trichoplusia ni)幼虫,且HBsAg基因在草地夜蛾(Spodoptera frugiperda)离体细胞中的表达量要比前者高约37％,在虫体中的表达则更高。     

一株高水平表达重组蛋白昆虫细胞系的建立

报道了一株来自粉纹夜蛾Trichoplusiani脂肪体的传代细胞系 ,在辅以 5%胎牛血清的商品无血清培养基Excell 4 0 0中 ,细胞群体倍增时间为 2 2 9h ,最高密度可达 2 2× 10 6 mL ,该细胞对苜蓿丫纹夜蛾多粒包埋型多角体病毒 (AcMNPV)极为敏感 ,增殖AcMNPV多角体平均每个细胞达86个 ,表达由AcMNPV构建的重组蛋白的水平较高 ,β 半乳糖苷酶的表达水平为 ( 2 2 5 5± 13 4 )IU mL ;碱性磷酸酶的表达水平为 ( 4 7± 0 61)IU mL ,是一株高水平表达重组蛋白的传代细胞系 ,命名为HNU Tn FB1。     

Effects of infection with a granulosis virus on larval growth, development and ecdysteroid production in the cabbage looper, Trichoplusia ni

ABSTRACT. Granulosis virus-infected Trichoplusia ni (Hûbner) larvae exhibited an increased larval life span with no supernumerary moult and no pupation. Weight gain was not affected. Insects infected shortly after hatching were slower in reaching the fourth and fifth stadia than were control insects. Haemolymph ecdysteroid titres were lower in virus-infected insects than control insects, but these differences were only significant ( P <0.05) in the fifth stadium. Electron microscopic examination of the pro thoracic glands revealed extensive granulosis virus infection, and glands from virus-infected insects produced no RIA-detectable ecdysteroids in vitro. Injection of 20-OII-ecdysone into virus-infected larvae at various concentrations and times did not induce pupation.     

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: Large scale production of the E1 protein

The two envelope glycoproteins of rubella virus (RV), El of 58 kDa and E2 of 42–47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10−1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.     

Insect Diversity in Phytoremediation and Bioaccumulation of Se

A variety of plant species are being considered for the phytoremediation of selenium (Se) contaminated soils in agricultural regions of central California. Use of this plant-based technology may also attract a wide range of insects to these Se-accumulating plants. The first field study surveyed the diversity of insects attracted to tall fescue, birdsfoot trefoil, kenaf, and Indian mustard. Over 7500 specimens were collected by a sweep net collection technique for one complete growing season. Most of the 84 families identified were associated with beneficial insects, although pestiferous insects, for example, thrips, aphids, lygus, and leafhoppers, were also found. In the second study the bioaccumulation of Se in the cabbage looper [Trichoplusia ni (Hübner)] was investigated on Indian mustard grown in Se-rich water culture solution. Neonate larvae were transferred to plants and fed on Se-treated and no Se treated plants (controls) for 14 days, respectively. Pupae were collected from each treatment and incubated until adult insects emerged. Almost 50% fewer pupae were collected from Se-treated plants compared with “controls”, resulting in fewer adult insects. Selenium concentrations were as high as 3173 μg Se kg^-1 DW in adult insects hatched from Se-treated plants compared with <5 μg Se kg^-1 DW in insects from “controls”. Based on both studies, we concluded that insect diversity should be determined and insects monitored for bioaccumulation of Se on phytoremediation sites in agricultural regions.