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.     

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。     

粉纹夜蛾颗粒体病毒重组增效蛋白的增效作用

采用时间 剂量 死亡率模型 ,分析了粉纹夜蛾 (Trichoplusiani)颗粒体病毒重组增效蛋白P96对棉铃虫 (Helicoverpaarmigera)核型多角体病毒 (HaNPV)感染棉铃虫幼虫的增效作用。结果显示 :感染后 11d ,HaNPV P96组的LC50 值为 3.4 7× 10 3 多角体 /mL ,比HaNPV组 ( 3.89× 10 4 多角体 /mL)降低了 91.0 8% ;在 1.6× 10 4 ～ 1.6× 10 6多角体 /mL浓度范围内 ,HaNPV P96组的LT50 值较HaNPV组缩短 0 .3～ 1.8d。P96显著提高了HaNPV对棉铃虫幼虫的毒力     

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。     

HaSNPV几丁质酶基因在大肠杆菌和昆虫细胞中的表达及其产物对Bt杀虫剂的增效作用

为了探索杆状病毒几丁质酶对微生物杀虫剂的增效作用及其利用途径 ,分别在大肠杆菌和昆虫细胞中表达棉铃虫单粒包埋型核型多角体病毒 (HaSNPV)几丁质酶 .用PCR方法扩增出不含N端信号肽编码序列的几丁质酶基因片段 ,并分别克隆至原核表达载体pET2 8a和重组到杆状病毒BactoBac表达系统 ,在大肠杆菌 (E .coli)BL2 1和粉纹夜蛾 (Trichoplusiani)细胞系Tn 5B1 4中分别进行了表达 .在大肠杆菌中表达量约占细菌总蛋白 15 % ,在昆虫细胞中表达量约占细胞总蛋白10 % .将含有几丁质酶的大肠杆菌和昆虫细胞表达产物添加到苏云金杆菌 (Bt)菌液中一起喂食 2龄家蚕 .结果显示 ,HaSNPV几丁质酶基因的 2种表达产物和Bt杀虫剂的混合物使处理的家蚕的致死时间较对照处理均明显缩短 .昆虫细胞和大肠杆菌表达产物与Bt混合物处理的LT50 分别从 93 5h和 95 1h缩短到 5 6 2h及 6 7 2h ,并且供试家蚕的生长速度明显缓慢 .研究结果表明 ,重组的HaSNPV几丁质酶有望作为Bt杀虫剂的增效剂     

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.     

真菌新型激活蛋白对Bt制剂的增效作用

激活蛋白是从交链孢属 (Alternaria)真菌分离的新型多功能蛋白 ,以棉铃虫和小菜蛾幼虫为供试昆虫 ,用饲料染毒法、浸叶法和浸虫法测定了激活蛋白对Bt的增效作用。结果表明 ,用Bt∶激活蛋白为 1∶0 0 3的含毒饲料饲喂 2龄棉铃虫幼虫 ,其增效指数为 2 2倍。Bt∶激活蛋白以 1∶0 0 95和 1∶0 6混合后分别浸叶后 ,饲喂棉铃虫和小菜蛾幼虫 ,其增效指数分别为 2 7和 5 5倍。单独用激活蛋白饲喂棉铃虫幼虫无致死活性 ,但当Bt与激活蛋白以 1∶0 75混合后 ,毒杀效果显著增强 ,对Bt的增效指数为 18 5倍。     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini-F replicon、一个卡那霉素抗性基因和一个lacZα基因8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内,构建了既能在E.coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid-HZ8).另外将HaSNPV的多角体蛋白基因和P10启动子序列取代pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列,构建插入HaSNPV多角体蛋白基因和 P10启动子序列的HapFastBacPhP10供体质粒.利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上,随后将含有eGFP基因的重组HaBacmid DNA转染至HZAm1细胞内.转染5d后,细胞核内能形成典型的多角体,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光.结果证明我们构建的HaBac to Bcac 表达系统能有效的表达外源基因.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.