粘虫颗粒体病毒的增效因子提高杆状病毒的感染

一株美洲粘虫GV(增效品系)在东方粘虫幼虫上进行增殖,所获粘虫GV(PuGv-Ps>对东方粘虫NPV(PsNPV)、棉铃虫NPV(HaNPV)和黄地老虎NPV(AsNPV)进行增效试验。 实验结果证明PuGV-Ps对三种NPV都有明显的增效作用, 其中以对PsNPV为最强,增效率达55%一85%;对HaNPV为30%-80%;而对AsNPV只有15%-35%。用凝胶过滤技术从PuGV-Ps中分离增效因子PusF-Ps,其对PsNPV和A,NPV的增效作用亦同样明显,250μg的PuSF-Ps可以提高PsNPV的感染能力达80%。经ScPhadcf-150柱(1.6X 90)和使用四种标准蛋白(细胞色素c、胃蛋白酶、牛血清清蛋白和乳酸脱氢酶)测得PuSF-Ps的分子量为160 000左右。     

粘虫颗粒体病毒增效因子的分离纯化及其生化性质

粘虫颗粒体病毒经０．０２ｍｏｌ／ＬＮａＯＨ碱溶,先用ＳｅｐｈａｄｅｘＧ－２００凝胶过滤层析柱从病毒蛋白粗提中分离增效因子,然后选用ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析柱进一步纯化增效因子,得到少量电泳纯的增效因子蛋白样品。     

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。     

东方粘虫颗粒体病毒超氧化物歧化酶基因的克隆与分析

为获得东方粘虫颗粒体病毒(Pseudelatia separata granulovirus,PsGV)基因组序列,采用随机克隆方法,建立PsGV的质粒基因文库,并通过对插入片段进行克隆鉴定和序列分析,获得编码超氧化物歧化酶蛋白的基因(PsGV-sod)。该基因阅读框为462bp,共编码153个氨基酸。核苷酸和氨基酸同源性比较结果表明该基因与其他颗粒体病毒同源性较高,通过保守基序分析,认为其为铜锌超氧化物歧化酶。     

黄地老虎颗粒体病毒DNA片段的克隆与颗粒体蛋白基因的定位

用ｐＵＣ１９质粒作载体,克隆了黄地老虎颗粒体病毒（Ａｇｒｏｌｉｓｓｅｇｅｔｕｍｇｒａｎｕｌｏｓｉｓｖｉｒｕｓ,简称ＡｓＧＶ）ＤＮＡＰｓｔＩ－Ｄ．Ｅ．Ｆ．Ｇ．Ｈ．Ｊ．Ｋ．等７个片段。以［￣（３２）Ｐ］－ｄＣＴＰ标记的油桐尺蠖核型多角体病毒（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａｎｕｃｌｃａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ简称ＢｓＮＰＶ）多角体蛋白基因为探针,在３７℃条件下对ＡｓＧＶ）颗粒体蛋白基因进行了定位,将其分别定位于ＢｓｌⅡ－Ｓ或ＴＰｓＴＩ－Ａ或Ｂ和ＥｃｉＲＩ－Ａ片段上。     

粘虫核型多角体病毒和颗粒体病毒侵染粘虫前胸腺的病理观察

观察比较了粘虫核型多角体病毒(Pseudelatia separata NPV,PsNPV)、粘虫颗粒体病毒(Pseudelatia separataGV,PsGV)感染粘虫,以及两种病毒混合感染粘虫后,粘虫(Pseudelatia separata)前胸腺的形态特征和前胸腺细胞的超微结构.结果表明,不同感染组粘虫前胸腺腺体都有不同程度的组织病变,PsNPV感染组在感染晚期与前胸腺相连的气管严重病变,出现大量白色颗粒状物累积,被伊红染成紫黑色,腺体细胞被挤压变形;PsGV感染组的前胸腺腺体变小,单个细胞也小,细胞界限不十分明显;两种病毒混合感染组的腺体细胞小,能被伊红染色的细胞极少.同时,前胸腺细胞的超微结构也有不同程度的病变.     

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...     

粘虫核型多角体病毒和颗粒体病毒侵染粘虫前胸腺的病理观察

观察比较了粘虫核型多角体病毒(Pseudelatia separata NPV,PsNPV)、粘虫颗粒体病毒(Pseudelatia separata GV,PsGV)感染粘虫,以及两种病毒混合感染粘虫后,粘虫(Pseudelatia separata)前胸腺的形态特征和前胸腺细胞的超微结构。结果表明,不同感染组粘虫前胸腺腺体都有不同程度的组织病变,PsNPV感染组在感染晚期与前胸腺相连的气管严重病变,出现大量白色颗粒状物累积,被伊红染成紫黑色,腺体细胞被挤压变形;PsGV感染组的前胸腺腺体变小,单个细胞也小,细胞界限不十分明显;两种病毒混合感染组的腺体细胞小,能被伊红染色的细胞极少。同时,前胸腺细胞的超微结构也有不同程度的病变。     

中国棉铃虫核型多角体病毒几丁质酶基因的定位与克隆

以α３２ＰｄＡＴＰ标记含ＣｆＭＮＰＶ几丁质酶基因的重组质粒为探针,在６８℃条件下对棉铃虫单粒包埋核型多角体病毒（ＨａＳＮＰＶ）进行Ｓｏｕｔｈｅｒｎ杂交,将ＨａＳＮＰＶ的几丁质酶基因分别定位在ＢａｍＨＩＥ、ＢｇｌⅡＥ、ＥｃｏＲＩＧ、ＨｉｎｄⅢＦ、ＸｂａＩＨ、ＢａｍＨＩ＋ＨｉｎｄＩＩＭ和ＢａｍＨＩ＋ＸｂａＩＨ,并以ｐＴＺ１９Ｒ为载体获得了ＸｂａＩＨ片段克隆。     

粘虫颗粒体病毒对苏云金杆菌的增效特性及对Bt毒蛋白的降解活化作用

以小菜蛾Plutella xylostella为试虫,采用生物测定方法测定了粘虫颗粒体病毒（Pseudaletia unipuncta granulosis virus,PuGV-Ps）对苏云金杆菌（Bacillus thuringiensis,Bt）的增效作用。结果表明：不同配伍PuGV-Ps和Bt间的共毒系数在105.3至195.0之间, PuGV-Ps对Bt毒力具有增强作用,其中以Bt∶PuGV-Ps为4∶1增效作用最明显,72 h LC₅₀为0.039 mg/mL。不同温度和pH值都影响PuGV-Ps对Bt的增效作用,16℃～20℃增效程度明显高于24℃～32℃,而碱性条件下（pH 8～9）增效作用更显著。PuGV-Ps对Bt的增效作用因小菜蛾龄期不同而变化,2、3龄幼虫死亡率较单独使用Bt分别提高了50%和30.31%,而作用于低龄（1龄）和高龄（4龄）幼虫时对Bt的增效作用不显著。PuGV-Ps饲喂2 h后再接毒Bt,小菜蛾死亡率明显提高,48 h死亡率达66.67％,较直接饲喂Bt+PuGV-Ps处理死亡率提高了53.87％,差异极显著。SDS-PAGE表明PuGV-Ps具有碱性蛋白酶的活性,离体条件下能促进δ-内毒素酶解为47 kD,60 kD和61 kD的毒性肽。     

Phospholipid,an enhancing component in the synergistic factor of a granulosis virus of the armyworm,Pseudaletia unipuncta

The synergistic factor (SF) in the capsule of a granulosis virus (Hawaiian strain) of the armyworm, Pseudaletia unipuncta, contained polypeptides and phospholipids. Its molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 126,000 ± 8,700. The capsule proteins were digested by a proteinase released from the capsule under alkaline conditions, and by trypsin added to the proteinase-free capsules. Neither enzyme affected the synergistic factor or its activity. The synergistic factor was slowly depolymerized by 2% sodium dodecyl sulfate and was more rapidly depolymerized when phospholipase C (phosphatidylcholine cholinephosphohydrolase) was also added. Phospholipase C alone did not decompose the synergistic factor, but it did destroy the capacity of the synergistic factor to enhance the nuclear polyhedrosis virus. In contrast, phospholipase A₂ (phosphatidyl 2-acylhydrolase) had no effect on the synergistic factor. The different reactions of the two phospholipases on the synergistic factor suggested that the hydrophilic group of the phospholipid was exposed to the action of phospholipase C and was associated with the synergistic activity. This interpretation was supported by the detection of a phospholipid in the SF by thin-layer chromatography.     

Responsiveness of Pseudaletia unipuncta males to the female sex pheromone

ABSTRACT. In wind tunnel experiments male response to different concentrations of synthetic pheromone varied with age. At 25°C responsiveness increased up to day 5 and then declined slightly on day 7. For any given age the level of response generally increased with an increase in pheromone concentration. Males held at 15°C (since emergence) showed a continual increase in responsiveness with age; however, overall response levels were less than at 25°C. At 25°C male response to low concentrations of pheromone (10 and 30 μg) varied markedly over a 24 h period but no differences were observed at higher concentrations (100 and 300 μg).     

Location of a synergistic factor in the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SyF), which enhanced the infection of nuclear polyhedrosis viruses, was purified from capsules of a Pseudaletia unipuncta granulosis virus (Hawaiian strain) by immune affinity chromatography. The isolated SyF consisted primarily of a protein with molecular mass 98 kDa. The recovery rate depended on the alkali used to dissolve the capsules: the highest rate occurred with 0.05 M Na2CO3-0.05 M NaCl, followed in turn with 0.02-0.05 M NaOH and 0.04 M NaOH-0.05 M glycine. The solubilized components from untreated capsules contained 98- and 100-kDa proteins in addition to the matrix protein (29 kDa) and its decomposed products, while those from heat-treated capsules contained only the 100-kDa protein. Virons liberated from the capsules with the glycine buffer contained three proteins (33, 98, and 100 kDa) serologically related to the SyF. Immunoelectron microscopy of infected tissue and purified virions revealed the localization of the SyF antigens on the viral envelope.     

粘虫颗粒体病毒与苏云金杆菌联合作用对甜菜夜蛾的影响

以甜菜夜蛾为试虫,测定了粘虫颗粒体病毒（PuGV-Ps）对苏云金杆菌（Bt）毒力的增效作用。结果表明PuGV对甜菜夜蛾没有致毒作用,但Bt中加入PuGV后可以提高Bt对甜菜夜蛾的毒力,甜菜夜蛾致死中量LC50由Bt单剂的1．094mg／mL下降到0．862mg／mL,共毒系数达127。亚致死剂量Bt处理甜菜夜蛾影响了幼虫的生长发育,表现为幼虫生长量相对减少、蛹重下降、化蛹率降低和化蛹历期延长,添加了PuGV-Ps后进一步增强了Bt对甜菜夜蛾的生长发育的抑制作用。甜菜夜蛾中肠蛋白酶活性测定结果表明,PuGV-Ps对甜菜夜蛾中肠酶活性具有抑制作用;昆虫同时取食PuGV-Ps和Bt后,中肠酶液总蛋白酶活力都有所下降,在中肠酶液最适pH范围内蛋白酶活力抑制作用最明显。     

Serological study on the transmission of a granulosis virus of the armyworm,Pseudaletia unipuncta (Lepidoptera: Noctuidae), to other lepidopterous species

The synergistic (Hawaiian) strain of a granulosis virus (GV) from the armyworm, Pseudaletia unipuncta, was transmitted to Spodoptera exigua, Autographa californica, and Trichoplusia ni. The viruses isolated from these hosts were tested by radial double-immunodiffusion (RDD) and immunoelectrophoresis (IE) for their relationship to the original virus. Untreated and heat-treated virus inclusion bodies (capsules) were compared for their antigenic properties but no differences were detected. The antiserum elicited against the whole capsule was more sharply specific for the antigenic determinants than the one elicited against the dissolved capsule proteins. The viruses obtained from S. exigua and T. ni elicited precipitin lines that differed from those of the P. unipuncta GV in their electrophoretic mobility with the one-trough IE method; however, with the two-trough method, the lines fused indicating that the antigens were identical. The major precipitin line indicating identity of the viruses wasthat produced by the synergistic factor (SF) purified from the capsule proteins of the synergistic GV strain. The presence of SF in the GV produced in alternate hosts indicated that its production was virus directed. The SF was not detected in the GVs of Laspeyresia pomonella and Pieris rapae and in the nonsynergistic Oregonian GV of P. unipuncta. A field-collected GV from S. exigua exhibited a different precipitin pattern from that of the synergistic GV, but one of the precipitin lines shared partial relationship to the SF.     

Site of action of a synergistic factor of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SF), which is present in the capsule matrix protein of a granulosis virus of the armyworm, Pseudaletia unipuncta, enhances baculovirus infection in armyworm larvae. The site of action of the SF was investigated. The oral inoculation of SF did not enhance the infectious hemolymph virions which had been inoculated into the hemocoel. The SF also did not enhance the infection of purified enveloped virions when both virus and SF were inoculated into the hemocoel, but enhancement occurred when they were inoculated orally. Thus, the activity of the SF was confined to the midgut lumen. Observations with ferritin-conjugated antibody indicated that the site of action of SF was the cell membrane of the microvillus. There were more ferritin particles attached to midgut cell membranes of larvae inoculated orally with SF than to those of control larvae inoculated with buffer.