重组病毒杀虫剂应用研究进展

应用分子生物学技术可以将昆虫特异性的毒素基因、某些酶基因等外源基因插入昆虫病毒基因组,或通过改造昆虫病毒基因组等方法构建重组病毒杀虫剂,提高杀虫效果。温室及田间释放实验证实,重组病毒杀虫剂可以显著提高现场防治效果。连续多代抗性筛选实验表明,宿主被诱导产生对重组病毒杀虫剂抗性的速度低于野生型病毒杀虫剂。采用在剂型中添加光增白剂等保护剂、在基因组中插入具有增效作用的基因、应用病毒增强蛋白等技术可以提高重组病毒杀虫效果。随着基因工程技术的发展和安全性研究的深入,以重组杆状病毒为主的重组昆虫病毒杀虫剂的应用研究正面临着突破。     

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.     

昆虫杆状病毒几丁质酶及其应用研究进展

杆状病毒几丁质酶基因（chitinase,,ChiA）是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区：N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原（pro-V-Cath）的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiA或V-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。     

生物工程增效蛋白复合生物杀虫剂

武汉大学生命科学学院病毒研究所科研工作者,近年研究出了一种新的生物杀虫剂,即生物工程增效蛋白复合生物杀虫剂,也即是高效广谱病毒(CPV)。 这种病毒杀虫剂是从国内首次筛选出的败血型病毒与我国首次研制的增效工程蛋白,按一定比例组配研制而成。这种病毒性生物杀虫剂是将颗粒体病毒的增效蛋白基因克隆于表达载体,并进一步在大肠杆菌内再表达增效而制成的工程蛋白,有较强的杀虫能力,能显著提高病毒在虫体的感染力和协同杀虫效果好的苏云金杆菌的毒力。与此同时,它还是微生物杀虫剂的中间体,可广泛用于病毒杀虫剂、Bt杀虫剂和转Bt基因棉的杀虫剂。其应用范围很广,可对农、林、牧等鳞翅目的主要害虫,尤其对夜蛾科害虫有特效。例如斜纹夜蛾、银纹夜蛾、粉纹夜蛾、甜菜夜蛾以及菜青虫、棉铃虫、松毛虫等。这说明它杀虫谱广。同时,杀虫速度快,可造成“靶”昆虫幼虫瘫痪死亡。因其毒力破坏了靶昆虫幼虫肠道的微食膜,所以造成害虫停食、停止运动并最后死亡。 据统计,我国棉田(地)约5000万亩,森林9亿亩,蔬菜400万亩。危害棉花、蔬菜和森林的棉铃虫、松毛虫和夜蛾科的害虫繁殖快,危害严重和抗药性强,若用传统农药,效果不十分显著,现采用生物工程增效蛋白复合生物杀虫剂,可确保农林牧增产和丰收。秦春圃     

核衣壳蛋白VP39融合TAT转导肽对杆状病毒转导哺乳动物细胞的影响

为了提高昆虫杆状病毒在哺乳动物细胞中转导基因的效率,构建了重组杆状病毒AcRed-tat和AcRed。两者都能在哺乳动物细胞内表达红色荧光蛋白作为报告基因。同时,AcRed-tat带有HIV-1Tat转导肽、病毒主要衣壳蛋白基因vp39及增强型绿色荧光蛋白(egfp)三者的融合基因,并由杆状病毒多角体启动子表达,能够在昆虫细胞中表达该Tat融合蛋白,并掺入子代病毒粒子。而AcRed作为相应的对照病毒,带有多角体启动子表达vp39和egfp的融合基因。2株病毒分别转导哺乳动物细胞后,利用流式细胞仪检测报告基因的表达水平,发现在CHO和Vero细胞中AcRed-Tat介导的报告基因表达水平明显高于AcRed,而在HEK293细胞中2株病毒介导的报告基因表达水平差异不显著。结果表明Tat转导肽可以提高杆状病毒对一部分哺乳动物细胞的转导效率,为改进杆状病毒-哺乳动物细胞转导载体提供了新的思路。     

昆虫病毒gp37/fusolin基因研究进展

昆虫病毒gp37fusolin基因属病毒复制非必需基因,其编码的蛋白能形成一种纺锤形包涵体(spindlebodies,SBs),该包涵体内不含有病毒粒子。Fusolin蛋白形成的SBs与纯化的Fusolin蛋白均能提高靶昆虫对杆状病毒的敏感性,推测GP37蛋白也具有类似的功能。对gp37fusolin基因的深入研究,有可能开发出新型病毒增效剂 。     

杆状病毒SpltMNPVSl136基因的克隆、表达及其产物功能

计算机分析斜纹夜蛾核多角体病毒（SpltMNPV)基因组序列,发现第136个读码框基因表达产物具有病毒囊膜蛋白的基本特征,计算机预测的SL136蛋白氨基酸序列N端具有信号肽,C端具有跨膜区,N端区还有一个许多病毒融合蛋白共有的卷曲螺旋结构。通过PCR扩增,我们克隆了Sl136基因并分别构建了原核表达载体pBVSl136及重组病毒表达载体,SDS-PAGE结果表明Sl136基因在大肠杆菌和昆虫细胞中均获得了较高的表达,另外,我们还构建了一个瞬时表达载体pUCSl136。单独转染Sl-zsu-1细胞后,低pH值环境可诱导细胞发生膜融合并形成合胞体,这些实验结果表明,Sl136基因表达的产物是一个病毒膜融合蛋白。     

杆状病毒介导外源基因在哺乳动物细胞中表达的研究进展

杆状病毒(Baculovirus)是一种以昆虫为唯一宿主的病毒, 可用做生物杀虫剂或作为表达载体在昆虫细胞中大量表达外源蛋白, 制备疫苗。研究发现, 在哺乳动物细胞中携带哺乳动物启动子的重组杆状病毒能启动下游外源基因的表达但病毒不能在哺乳动物细胞中增值, 对细胞毒性小, 转导成功的细胞可以稳定传代并有效表达外源基因, 哺乳动物细胞比昆虫细胞对蛋白质具有更好的翻译后修饰, 表达出的蛋白结构更接近天然蛋白。因此, 杆状病毒可作为一种新型的哺乳动物细胞基因转移载体, 用于表达外源基因及作为一种基因治疗载体, 具有巨大潜力, 日益受到人们的关注。本文对杆状病毒作为一种表达载体在哺乳动物细胞中表达的研究进展进行了综述。     

增强蛋白与多角体蛋白共包埋的研究

颗粒体病毒的增强蛋白（enhancin)是一种能显著提高核型多角体病毒（NPV）对昆虫感染力的病毒蛋白。构建了一种不形成多角体但表达粉纹夜蛾颗粒体病毒增强蛋白的重组病毒AcBBH-TnEn,将它与野生型AcMNPV共同感染SF21细胞,经SDS－PAGE、免疫印迹分析、荧光免疫等方法检测证实,增强蛋白与多角体可在同一细胞中同时表达,而且发现所形成的病毒多角体带有增强蛋白。这表明,可以通过混合感染的方式生产带有增强蛋白的病毒多角体。     

具有生物安全性的杆状病毒杀虫剂基因工程技术的发展与前景

杆状病毒作为杀虫剂在世界各地已被广范应用．但与化学农药相比,杆状病毒具有杀虫速度慢,对高龄害虫需用量大,杀虫谱窄等缺点．随行基因工程技术的发展,从80年代末期起,科学家开始尝试对杆状病毒的遗传性状进行各种分子生物学改造,以获得更优良的病毒虫剂。近年来这方面的研究已取得了可喜进展,同时重组病毒的安全性也引起了世界范围的广泛关注。因此．研制既有优良杀虫性能,又有生物安全性的重组病毒．已成为当今病毒杀虫剂的发展方向。本文及时地总结了重组杆状病毒杀虫剂研究的历史,从提高病毒杀虫速度、增强病毒杀虫毒性、以及病毒宿主特异性等三个方面进行了系统归纳,并对重组病毒的安全性进行科学地分析。重点阐述了新时期研制具有生物安全性的重组病毒的各项基因工程策略,如对重组病毒进行混合包装、前包装：或生产缺陷型的重组病毒（p10基因,p74基因．egt基因,pp34基因的缺失）等。这些措施将把重组病毒对环境可能造成的危害控制在最小范围。理想的重组病毒杀虫剂应具有杀虫快、杀虫谱广、不危害其它生物、在环境中滞留时间短等特点。     

The Lymantria dispar nucleopolyhedrovirus enhancins are components of occlusion-derived virus

Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of approximately 84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.     

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     

Comparison of the bacterial Enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin

Enhancins are a class of metalloproteases found in some baculoviruses that enhance viral infection by degrading the peritrophic membrane (PM) of the insect midgut. However, sequencing has revealed enhancin-like genes with 24-25% homology to viral enhancins, in the genomes of Yersinia pestis and Bacillus anthracis. AcMNPV does not encode enhancin therefore recombinant AcMNPV budded viruses (BVs) and polyhedra inclusion bodies (PIBs) were generated expressing the bacterial Enhancins. Bacterial Enhancins were found to be cytotoxic when compared to viral enhancin, however, larval bioassays suggested that the bacterial Enhancins did not enhance infection in the same way as viral Enhancin. This suggests that the bacterial Enhancins may have evolved a distinct biochemical function.     

Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

High-throughput analysis of rat liver plasma membrane proteome by a nonelectrophoretic in-gel tryptic digestion coupled with mass spectrometry identification

In-gel digestion is commonly used after proteins are resolved by polyacrylamide gel electrophoresis (SDS-PAGE, 2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the direct analysis of complex proteins. Here, we describe a strategy combining isolation of purified plasma membrane, efficient digestion of plasma membrane proteins in polyacrylamide gel, and high-sensitivity analysis by advanced mass spectrometry to create a new rapid and high-throughput method. The plasma membrane protein mixture is directly incorporated into a polyacrylamide gel matrix, After formation of the gel, proteins in the gel section are digested with trypsin, and the resulting peptides are subjected to reversed-phase, high-performance liquid chromatography followed by electrospray ion-trap tandem mass analysis. Using this optimized strategy, we have identified 883 rat liver membrane proteins, of which 490 had a gene ontology (GO) annotation indicating a cellular component, and 294 (60%) of the latter were known integral membrane proteins or membrane proteins. In total, 333 proteins are predicted by the TMHMM 2.0 algorithm to have transmembrane domains (TMDs) and 52% (175 of 333) proteins to contain 2-16 TMDs. The identified membrane proteins provide a broad representation of the rat plasma membrane proteome with little bias evident due to protein p I and molecular weight (MW). Also, membrane proteins with a high GRAVY score (grand average hydrophobicity score) were identified, and basic and acidic membrane proteins were evenly represented. This study not only offered an efficient and powerful method in shotgun proteomics for the identification of proteins of complex plasma membrane samples but also allowed in-depth study of liver membrane proteomes, such as of rat models of liver-related disease. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of rat liver plasma membrane in general.     

The role of lipids in the biogenesis of integral membrane proteins

Most integral membrane proteins are cotranslationally inserted into the lipid bilayer. In prokaryotes, membrane insertion of the nascent chain takes place at the plasma membrane, whereas in eukaryotes insertion takes place into the endoplasmatic reticulum. In both kingdoms of life, however, the same membrane that acquaints the newly born membrane protein also synthesizes the bilayer lipids and thus ensures the balanced growth of the membrane as a whole. Recent evidence indicates that the lipid composition of the host membrane can determine the fate of the newborn membrane protein, as it can affect (1) the efficiency of translocation, (2) the topology of the resulting membrane protein, (3) its stability, (4) its assembly into oligomeric complexes, (5) its transport and sorting along the secretory pathway, and (6) its enzymatic activity. The lipid composition of the membrane thus can affect the biogenesis and function of integral membrane proteins at multiple steps along its biogenetic pathway. While understanding this interdependence between bilayer lipids and protein biogenesis is interesting in its own right, careful consideration of a potential host’s membrane lipid composition may also allow optimization of the yield and activity of membrane proteins that are expressed in a heterologous organism. Here, we review and discuss some examples that illustrate the interdependence between bilayer lipids and the biogenesis of integral membrane proteins.