利用超氧化物歧化酶增加棉铃虫病毒的感染性

棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)是棉铃虫的专一性病原,具有宿主特异性高、不影响天敌、不污染环境、对人畜安全等优点,在我国作为商品生物杀虫剂已有10余年的历史.     

杆状病毒凋亡抑制基因

杆状病毒(baculoviruses)感染昆虫细胞会引发细胞凋亡,然而病毒为了确保自身的复制和繁殖会抑制宿主细胞的凋亡.杆状病毒在长期进化过程中获得了凋亡抑制基因,如苜蓿银纹夜蛾核型多角体病毒(Autographa californica MNPV, AcMNPV)中的p35基因,棉铃虫核型多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus, HaSNPV)基因组中的IAP基因家族,以及莲纹夜蛾核形多角体病毒(Spodoptera littoralis multicapsid nucleopolyhedrovirus, SpliMNPV)的p49基因等.尽管这些基因都具有抑制细胞凋亡的功能,但是作用途径和方式却各有差异.对杆状病毒3种抗凋亡基因的结构和功能作一简单的介绍和评述.     

棉铃虫单粒包埋核型多角体病毒在细胞系中持续感染的建立和特性栽

棉铃虫单粒包埋核型多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)能够在棉铃虫蛹卵巢细胞素SFE-HA-8212中有效复制,引起细胞解体死亡.通过培养HaSNPV感染后残存的细胞,建立了一株持续感染的细胞.该细胞在传代培养的6个月时间内(约25次传代),持续地释放有感染力的病毒粒子,培养液中病毒的效价多在104 TCID50/ml上下,同时有一小部分细胞(1%左右)中形成多角体,释放病毒的细胞占总细胞数的1.0%～3.8%.持续感染细胞的生长特性与原细胞系相比有一定的变化,对HaSNPV感染的敏感性有所降低.另一方面,持续感染细胞所释放的病毒对SFE-HA-8212及棉铃虫幼虫的感染力也有所降低.讨论了持续感染的可能机制.     

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind Ⅲ-L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组的HindⅢ-L片段的全序列.该片段全长2 635bp,包括5个有意义的开放阅读框HaSNPV ORF227,晚期表达因子10基因(lef10),vp1054基因,Ac55(AcMNPV ORF55的同源基因),Ac56(AcMNPV ORF56的同源基因).与其它6种杆状病毒的氨基酸序列比较表明,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒(SeMNPV)的同源性最高,为64%,与冷杉毒蛾核型多角体病毒(OpMNPV)的同源性最低,为43%;HaSNPV的vp1054基因与SeMNPV的同源性最高,为65%,与OpMNPV的同源性最低,为49%.序列比较表明,HaSNPV的LEF10与VP1054蛋白与其它6种杆状病毒具有相同的保守区和亮氨酸拉链(1eucine zipper)     

棉铃虫病毒HaSNPV fp25k基因的克隆表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)fp25k基因的序列,设计引物,引入适当的酶切位点,利用PCR扩增出基因片段.将该基因片段克隆至原核表达镲载体pProEXHTb,经IPTG诱导,在大肠杆菌DH5α中获得了高效表达,表达产物的大小为32kDa.纯化蛋白产物免疫家兔制备抗血清.该抗血清可与原核表达的GST-FP25K融合蛋白及在感染的昆虫细胞中表达的FP25K蛋白发生特异性免疫反应.该抗体的获得为深入研究FP25K蛋白的功能提供了基础.     

SeMNPV抑制HaSNPV诱导的Se-UCR细胞凋亡

在采用共感染和共转染的方法构建扩大杀虫范围的重组病毒的研究过程中发现棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus, HaSNPV)能诱导甜菜夜蛾细胞Se-UCR发生典型凋亡,但不能诱导另一株甜菜夜蛾细胞Se-301产生凋亡.以5 MOI的HaSNPV感染Se-UCR,在12h左右可以观测到少量细胞凋亡,24h能观察到明显的凋亡,凋亡细胞数量随时间不断增加,到72h基本上所有的细胞均发生凋亡,成为凋亡小体,基因组DNA片段化.同时发现HaSNPV诱导的甜菜夜蛾Se-UCR细胞凋亡能够被甜菜夜蛾多核衣壳核多角体病毒(Spodoptera exigua multicapsid nucleoplyhedrovirus, SeMNPV)所抑制, 进一步点杂交试验发现SeMNPV 和HaSNPV共同感染Se-UCR获得了HaSNPV在该细胞中的复制.     

棉铃虫单核衣壳核多角体病毒（HaSNPV）Hind Ⅲ—L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒（Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV）基因组的HindⅢ－L片段的全序列。该片段全长2635bp,包括5个有意义的开放阅读框：HaSNPV ORF227,晚期表达因子10基因（lef10 ),vp1054基因,Ac55(AcMNPV ORF55的同源基因）,Ac56（AcMNPV ORF56的同源基因）。与其它6种杆状病毒的氨基酸序列比较表明,HaSNPV的lef10基因与甜夜蛾核型多角体病毒（SeMNPV）的同源性最高。为64％,与冷杉毒蛾核型多角体病毒（OpMNPV）的同源性最低,为43％;HaSNPV的vp1054基因与SeMNPV的同源性最高。为65％,与OpMNPV的同源性最低,为49％。序列比较表明,HaSNPV的LEF10与VP1054蛋白与其它6种杆状病毒具有相同的保守区和亮氨酸拉链（leucine zipper）。     

棉铃虫单粒包埋型核型多角体病毒(HaSNPV)复制的研究——Ⅰ.细胞病理学和病毒复制的一些特性

本文报道了棉铃虫单粒包埋型核型多角体病毒(Heliothis armigera single nucleocapsid nuclear polyhedrosis virus, HaSNPV)在棉铃虫及棉铃虫蛹卵巢细胞系SFE-HA-8212中的复制。HaSNPV的复制和其他的核型多角体病毒大体相符,复制过程也可分为形成出芽型病毒与形成包埋型病毒这两个时相。研究了影响病毒在细胞中复制的诸因素,包括病毒感染复数、细胞接种密度和细胞生长阶段等。在适宜的条件下平均每细胞可生产出芽型病毒14PFU,多角体24个。生成的病毒具有感染力。这些表明SFE-HA-8212细胞可供HaSNPV有效复制。同时,作为细胞群体该细胞系对HaSNPV感染的反应并非均一,其中有89.65±21.4％的感染细胞释放病毒,但仅有37.85±6.7％的细胞形成多角体。表明HaSNPV的感染并不一定导致形成多角体,在大部分感染细胞中病毒复制进行到产生病毒粒子就停止了。初步讨论了这种不均一性的原因。     

棉铃虫病毒HaSNPV fp25k基因的克隆表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)fp25k基因的序列。设计引物,引入适当的酶切位点,利用PCR扩增出基因片段。将该基因片段克隆至原核表达载体pProEXHTb,经IFTG诱导,在大肠杆菌DH5α获得了高效表达,表达产物的大小为32kDa。纯化蛋白产物免疫家兔制备抗血清。该抗血清可与原核表达的GST-FP25K融合蛋白及在感染的昆虫细胞中表达的FP25K蛋白发生特异性免疫反应。该抗体的获得为深入研究FP25K蛋白的功能提供了基础。     

SeMNPV抑制HaSNPV诱导的Se—UCR细胞凋亡

在采用共感染和共转染的方法构建扩大杀虫范围的重组病毒的研究过程中发现棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)能诱导甜菜夜蛾细胞,Se-UCR发生典型凋亡,但不能诱导另一株甜菜夜蛾细胞Se-301产生凋亡。以5MOI的HaSNPV感染Se-UCR。在12h左右可以观测到少量细胞凋亡。24h能观察到明显的凋亡,凋亡细胞数量随时间不断增加,到72h基本上所有的细胞均发生凋亡,成为凋亡小体,基因组DNA片段化。同时发现HaSNPV诱导的甜菜夜蛾Se-UCR细胞凋亡能够被甜菜夜蛾多核衣壳核多角体病毒(Spodoptera exigua multicapsid nucleoplyhedrovirus,SeMNPV)所抑制,进一步点杂交试验发现SeMNPV和HaSNPV共同感染Se-UCR获得了HaSNPV在该细胞中的复制。     

Field inactivation of wild-type and genetically modified Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus in cotton

Cotton bollworm (Helicoverpa armigera) is a serious pest on cotton in China. A specific baculovirus, H. armigera nucleopolyhedroviruses (HaSNPV) is used as a commercial biopesticide to control this pest. To improve the pesticidal properties, HaSNPV has been genetically engineered by both deleting the ecdysteroid UDP-glucosyltransferase (egt) gene from its genome (recombinant HaSNPV-EGTD) and incorporating an insect-selective toxin gene from the scorpion Androctonus australis (AaIT) (recombinant HaSNPV-AaIT). In the field, there was no significant difference among the inactivation rates of the two recombinant HaSNPVs and their parent wild-type, HaSNPV-WT. The inactivation rate of these viruses was significantly different in different years. The average half-life of HaSNPV was 0.57, 0.90 and 0.39 days in 2000, 2001 and 2002, respectively. Inactivation rates correlated well with solar radiation over these years.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.     

HaSNPV几丁质酶重组蛋白的表达、纯化和复性

棉铃虫单核衣壳核型多角体病毒（HaSNPV）是我国第一个商品病毒杀虫剂,具有使用安全、害虫不产生抗药性等优点,是一种很有发展潜力的生物农药。幼虫虫体受病毒感染后,HaSNPV几丁质酶在其液化过程中起了很大的作用,因此可以作为增效剂以显著提高细菌、病毒、真菌等微生物杀虫剂的毒力,并具有更高的安全性。将HaSNPV几丁质酶基因构建到原核表达载体pET28a中,经测序检验后转化至大肠杆菌Rosetta,然后以IPTG作为诱导剂,目标蛋白以包涵体的形式得以成功表达。在变性条件下,包涵体经镍 次氮基三乙酸(Ni-NTA)柱层析纯化,并以两种不同的方法进行复性,均可获得具有活性的HaSNPV几丁质酶。     

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind III-L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒 (Helicoverpaarmigerasingle nucleocapsidnucleopolyhedrovirus,HaSNPV)基因组的HindIII L片段的全序列。该片段全长 2 6 35bp ,包括 5个有意义的开放阅读框 :HaSNPVORF2 2 7,晚期表达因子 10基因 (lef10 ) ,vp10 5 4基因 ,Ac5 5 (AcMNPVORF5 5的同源基因 ) ,Ac5 6 (AcMNPVORF5 6的同源基因 )。与其它 6种杆状病毒的氨基酸序列比较表明 ,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒 (SeMNPV)的同源性最高 ,为6 4 % ,与冷杉毒蛾核型多角体病毒 (OpMNPV)的同源性最低 ,为 4 3% ;HaSNPV的vp10 5 4基因与SeMNPV的同源性最高 ,为 6 5 % ,与OpMNPV的同源性最低 ,为 4 9%。序列比较表明 ,HaSNPV的LEF10与VP10 5 4蛋白与其它 6种杆状病毒具有相同的保守区和亮氨酸拉链 (leucinezipper)     

AcMNPVP35抑制HaSNPV诱导的Tn-Hi5细胞凋亡

苜蓿银纹夜蛾核多角体病毒(Autographa californica multicapsid nuclear polyhedrosis virus,AcMNPV)能够抑制棉铃虫核多角体病毒(Helicoverpa armigera Nucleopoly hedrovirus,HaSNPV)诱导的Tn Hi5 细胞凋亡,并能辅助HaSNPV在Tn Hi5细胞中复制,产生具有感染能力的子代病毒。瞬时表达实验证明,在Tn Hi5细胞中,p35具有明显抑制凋亡的能力,但是不能辅助HaSNPV在Tn Hi5细胞中的复制;进一步构建超表达p35 的重组病毒:vHap35,发现vHap35能够抑制Tn Hi5细胞凋亡,但是不能产生具有感染力的病毒粒子。电镜观察发现感染重组病毒的部分细胞中存在单粒包埋的病毒粒子(ODV)。     

A new cell line from the embryonic tissue of Helicoverpa armigera HBN. (Lepidoptera: Noctuidae)

A new cell line from the embryonic tissue of Helicoverpa armigera was established and designated as NIV-HA-197. It was maintained in TNM-FH medium supplemented with 10% fetal bovine serum. The cell line at passage 20 had a heterogeneous population of cells consisting of mainly epithelial-like cells (70%), followed by fibroblast-like (27%), and multinucleated giant (3%) cells. The chromosome number ranged from 45 to 185. The growth curve at passage 40 showed a fivefold increase in cell number with a population-doubling time of approximately 60 h. The cell line was found infected with the microsporidium Nosema heliothids at passage 9. Using the antiprotozoan drug Metrogyl 400 and simultaneous heat treatment, the parasite was removed from the culture. The cell line can be cryopreserved for 30 mo. The species specificity of the new cell line was determined by studying the isoenzyme profile of four enzymes, viz., lactate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and glucose 6-phosphate dehydrogenase, and by heteroduplex analysis. Heteroduplex analysis was used to analyze the mitochondrial 16S ribosomal ribonucleic acid gene sequences along with the host insect gene sequences, and 100% homology was obtained, confirming the conspecificity of the cell line. The cell line was found to be susceptible to the baculoviruses Autographa californica multiple nucleopolyhedrovirus, Spodoptera litura multiple nucleopolyhedrovirus, and H. armigera single nucleopolyhedrovirus (HaSNPV). More than 90% of the cells were infected by HaSNPV on the seventh post infection day (PID), and 28.8 x 10(6) NPV/ml was yielded on the 10th PID. The in vitro-grown HaSNPV caused 100% mortality, when fed to the second instar H. armigera larvae, in 6 d. Cessation of feeding was observed on the second PID.     

中国棉铃虫核型多角体病毒不同基因型的虫体克隆

首次利用虫体克隆技术,对野生型中国棉铃虫核型多角体病毒（ＨａＳＮＰＶＷ）的不同基因型进行了分离纯化。以较低浓度的ＨａＳＮＰＶＷ感染三龄初中国棉铃虫幼虫,病毒致死幼虫单虫收集,分别提取相应的病毒核酸,进行限制性内切酶分析,得到了ＨａＳＮＰＶ的七个不同基因型（ＨａＳＮＰＶＧ１至Ｇ７）,其ＥｃｏＲＩ酶切图谱均不相同。用ＢａｍＨＩ酶切分析,七个基因型的酶切图谱只在一个片段上有显著区别：Ｇ１、Ｇ２、Ｇ３、Ｇ４ＤＮＡ的ＢａｍＨＩｈ片段大小是３．３０ｋｂ,Ｇ５、Ｇ６的这一片段的大小为３．５ｋｂ,而Ｇ７的这一片段为２．７ｋｂ。结果表明,野生型ＨａＳＮＰＶ至少包含七个不同的基因型。     

Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been developed as a commercial biopesticide to control the cotton bollworm, H. armigera, in China. The major limitation to a broader application of this virus has been the relative long time to incapacitate the target insect. Two HaSNPV recombinants with improved insecticidal properties were released in bollworm-infested cotton. One recombinant (HaCXW1) lacked the ecdysteroid UDP-glucosyltransferase (egt) gene and in another recombinant (HaCXW2), an insect-selective scorpion toxin (AaIT) gene replaced the egt gene. In a cotton field situation H. armigera larvae treated with either HaCXW1 or HaCXW2 were killed faster than larvae in HaSNPV-wt treated plots. Second instar H. armigera larvae, which were collected from HaCXW1 and HaCXW2 treated plots and further reared on artificial diet, showed reduced ST(50) values of 15.3 and 26.3%, respectively, as compared to larvae collected from HaSNPV-wt treated plots. The reduction in consumed leaf area of field collected larvae infected with HaCXW1 and HaCXW2 was approximated 50 and 63%, respectively, as compared to HaSNPV-wt infected larvae at 108 h after treatment. These results suggest that in a cotton field situation the recombinants will be more effective control agents of the cotton bollworm than wild-type HaSNPV.     

Efficient method to generate homologous recombinant baculovirus genomes in E. coli

Here we describe a convenient method to generate homologous recombinant baculoviral genomes in E. coli. The recombination takes place with the aid of recombination enzymes provided by the phage lambda Red system between a bacmid (a baculoviral genome that can replicate in bacteria) and a linear fragment. Proof of concept was provided when the cathepsin gene (v-cath) of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) was replaced by the chloramphenicol resistance gene (CmR). First, CmR was inserted between the flanking sequences of the HaS-NPV v-cath. Each of the flanking regions was about 1 kb. The fragment was linearized and electroporated into bacteria containing both the HaSNPV bacmid and the lambda Red system. Recombinant bacmids resistant to chloramphenicol were selected. In comparison to the standard co-transfection/plaque assays, this method significantly reduces the time required to construct baculovirus knockout mutants. It may also be useful in the manipulation of other large viral genomes.