培养基Grace＇s对棉铃虫细胞HzAm1培养的改进

昆虫细胞-杆状病毒表达体系给生物杀虫剂的工业化生产带来了希望。为了提高杆状病毒在昆虫细胞中的复制量并大规模生产生物杀虫剂,高性能、低成本培养基的选取十分必要。将Grace＇s培养基与IPL-41及Schneider＇s培养基的组分进行比较后,以Grace＇s为基础,向其中加入酵母提取物、水解乳蛋白、氨基酸以及钼、锰等微量元素进行改善,用于培养细胞HzAm1,最大细胞浓度提高了190.2%,改进效果明显。     

杆状病毒-昆虫细胞技术在人用重组蛋白疫苗生产中的应用

杆状病毒-昆虫细胞表达系统是生物领域四大表达系统之一。近年,随着Cervarix誖、Provenge誖和FluBlok誖的批准上市,杆状病毒-昆虫细胞表达系统的发展也取得了里程碑式的重大突破。同时,随着技术的快速发展,杆状病毒-昆虫细胞技术平台在疫苗研发和生产中的应用日益广泛而深入。本文拟对杆状病毒-昆虫细胞技术的起源、发展及其在人用重组蛋白疫苗生产中的应用作一综述。     

重组杆状病毒感染悬浮昆虫细胞感染策略优化及扩大试验

昆虫细胞-杆状病毒表达系统已经广泛应用于生物杀虫剂及重组蛋白的大规模生产。在昆虫细胞-杆状病毒表达系统中,感染策略中的感染复数(MOI)、感染时间(TOI)、细胞初始浓度(ICD)的相互关系还不是很清楚,而且相关报道也很少。本研究在摇瓶中利用正交实验探讨了MOI、TOI及ICD的相互作用关系以及对包涵体病毒(OV)和非包涵体病毒(BV)产量的影响。结果表明,感染复数对OV和BV产量有显著的影响;最佳感染条件为MOI为0.1,TOI为细胞处于对数生长期初期,ICD为2×105cells.ml-1;最大BV病毒滴度为2.0×107TCID50.ml-1。将在摇瓶中获得的最佳条件在气升式反应器进行扩大试验,获得最大病毒滴度为2.13×107TCID50.ml-1。     

昆虫细胞在预防性疫苗中的应用及外源因子的控制

昆虫细胞作为重组杆状病毒载体的宿主,广泛应用于重组蛋白的表达,在疫苗领域,昆虫细胞-杆状病毒表达系统的生产平台也越来越受到关注.但近年来随着科学技术的发展,多种昆虫细胞系被发现存在外源性病毒的持续感染,因此,昆虫细胞作为生物制品生产用细胞基质的安全性引起人们的密切关注.本文对杆状病毒表达系统中最常用的两种昆虫细胞SF9...     

人乳头瘤病毒结构蛋白L1与白色念珠菌甘露糖蛋白CTL表位嵌合基因在昆虫细胞中的表达

目的构建携带人乳头瘤病毒(HPV)结构蛋白L1和白色念珠菌甘露糖蛋白(CaMp65)细胞毒性T细胞表位(CTL)基因的重组质粒,并在杆状病毒-昆虫细胞系统中进行表达。方法分别设计包含HPV6bL1和CaMp65CTL145-153的引物,经PCR扩增嵌合基因HPV6bL1/CaMp65CTL145-153,并进一步将其插入杆状病毒表达载体pFastBacTM1。在E.coliDH10Bac中组装成重组杆状病毒,在昆虫细胞sf-9中表达嵌合蛋白,并经SDS-PAGE和Westernblot分析鉴定。结果HPV6bL1/CaMp65CTL145-153嵌合蛋白在昆虫细胞sf-9中得到了表达,表达产物的相对分子质量为56000,与HPV6bL1单抗能产生特异性反应。结论HPV6bL1/CaMp65CTL145-153嵌合蛋白在杆状病毒-昆虫细胞系统中得到了成功表达,为防治HPV和白色念珠菌感染的基因工程疫苗的研究奠定了基础。     

农用杀虫剂综述（上）

简单介绍了各类杀虫剂的主要研究成果及其发展，从化学杀虫剂包括有机磷杀虫剂、氨基甲酸酯杀虫剂、拟除虫菊酯杀虫剂、烟碱类杀虫剂、昆虫生长调节剂等，到生物杀虫剂包括微生物杀虫剂和植物杀虫剂等分别进行了综述性的报导。     

浙江大毒死蜱获中国专利金奖

近日,中科院武汉病毒研究所与江西宜春新龙化工有限公司签订技术合作协议,许可实施武汉病毒所发明专利“一种用替代宿主生产的广谱杆状病毒杀虫剂”,并在江西宜春建立生产线,生产广谱昆虫病毒甘蓝夜蛾核型多角体病毒杀虫剂。     

我国首次采用自主病毒分离提纯技术成功 昆虫病毒生物原药研发获突破

河南省济源白云实业有限公司成功建成我国最大的昆虫病毒生物杀虫剂原药生产基地。这些高品质昆虫病毒生物杀虫剂原药首次采用具有自主知识产权的棉铃虫群养技术和系统集成的病毒分离提纯技术，大幅度提高了产品的病毒含量，突破了严重制约该产品产业化发展的主要技术瓶颈，促进世界昆虫病毒生物杀虫剂产业跨上一个新台阶。     

武汉广谱昆虫病毒杀虫剂专利技术在江西实施生产

<正>近日,中科院武汉病毒研究所与江西宜春新龙化工有限公司签订技术合作协议,许可实施武汉病毒所发明专利"一种用替代宿主生产的广谱杆状病毒杀虫剂",并在江西宜春建立生产线,生产广谱昆虫病毒甘蓝夜蛾核型多角体病毒杀虫剂。     

Sf9细胞的高效扩增及类病毒颗粒高效自组装的培养工艺

目的优化SFX-Insect培养基及类病毒颗粒(virus like particles,VLPs)的自组装培养工艺,提高Sf9细胞的扩增效率及VLPs的自组装效率。方法以SFX-Insect培养基作为基础培养基,添加不同营养物(HyPep~(TM) 1510及葡萄糖),优化培养基。分别在摇瓶及WAVE生物反应器中培养Sf9细胞,同时补加不同倍数(1、2、5、8倍)的补料浓缩液,观察细胞培养效果;将重组戊型肝炎杆状病毒分别加入SFX-Insect及优化培养基培养的Sf9细胞中,分析目的蛋白表达量;根据细胞的不同生长阶段、接毒时是否补料及不同接毒MOI值,将优化培养基培养的Sf9细胞进行分组,分析各组细胞中目的蛋白的表达量并观察VLPs的形成。结果 SFX-Insect基础培养基中同时添加10 g/L葡萄糖和10 g/L HyPep~(TM)1510为Sf9细胞最佳培养基。优化培养基摇瓶培养Sf9细胞,活细胞密度高达2×10~7个/ml以上;在细胞对数生长中后期进行1及2倍补料可延长细胞平台生长期至20 d以上;将摇瓶培养的细胞放大至WAVE生物反应器中培养,密度高达107个/ml以上,且传代培养后,可维持10 d以上的平台生长期。优化培养基培养的Sf9细胞目的蛋白的表达量比SFX-Insect培养基提高了6倍;优化培养基培养Sf9细胞至对数生长中前期,进行补料接毒(MOI=2),VLPs的组装效率大幅提高,镜下可见形态均一的VLPs。结论成功优化了SFX-Insect培养基及VLPs的自组装培养工艺,提高了Sf9细胞的扩增效率及VLPs产量。     

In vitro maintenance of the sex pheromone gland of the female Indian meal mothPlodia interpunctella (Hübner)

Sex pheromone glands ofPlodia interpunctella were isolated from surface-sterilized donors of different ages, freed of most of the attached gut, oviduct, and other tissues; rinsed in sterile medium; and cultured in 1 ml of culture medium. The sex pheromone gland cells that were cultured for 10 days in either chemically defined Grace's medium or modified Grace's medium appeared normal in histological examinations. Bioassays of extracted medium in which pheromone glands from mature females had been incubated showed that a greater percentage of the available pheromone was recovered from modified Grace's medium than from chemically defined Grace's medium.Lepidoptera: Pyralidae.This research was supported in part by NIH Grant 1-F34 GM 06251.     

Insect cell cultivation techniques for the production of high-valued products

The large scale cultivation of insect cells will likely play an increasingly important role for the commercial production of bioproducts. While much research work has been done on the engineering aspects of insect cell suspension culture, many questions remain unanswered (i.e. the nature of bubble damage in sparged reactors, shear sensitivity and adaptation of cells; the use of serum-free medium). In addition, insect cell immobilization may become a suitable technique for enhancing cell densities and product concentrations. This article presents a brief critical review of the development of insect cell suspension culture techniques for the production of insect pathogenic viruses and recombinant proteins.The problems of culturing microencapsulated insect cells, infected with a temperature sensitive baculovirus, also is discussed.     

WAVE生物反应器在生物制品生产中的应用

WAVE生物反应器是一种新型的摇床式生物反应器,是新近发展起来的一种细胞培养的技术平台。其通过摇动板带动细胞袋内的培养基产生波浪,促进营养物质的交换和氧气的传递;细胞袋一次性使用,无需清洗和验证;具有悬浮培养和贴壁培养的功能,现已广泛应用于培养HEK293细胞、昆虫细胞、CHO细胞、杂交瘤细胞、NSO细胞、果蝇细胞、BHK细胞、植物细胞、杀伤性T细胞、MDCK细胞、EFL细胞、酵母菌等以及重组蛋白和单克隆抗体的生产。本文就WAVE生物反应器的组成、特点及其在生物制品生产中的应用作一综述。     

Humanization of lepidopteran insect-cell-produced glycoproteins

The insect cell-baculovirus expression vector system, widely used for glycoprotein production, is not ideal for pharmaceutical glycoprotein production due to the characteristics of the N-glycans in the expressed products. Insect cells lack several enzymes required for mammalian-type N-glycan synthesis and contain a specific N-acetylglucosaminidase that stunts the growth of chains and a core alpha-1,3-fucosyltransferase that yields potentially allergenic glycoforms. Current knowledge on N-glycan processing in lepidopteran insect cells is summarized, and strategies to develop better glycoprotein expression systems suitable for pharmaceutical glycoprotein production are discussed.     

Growth characteristics of a Bombyx mori insect cell line in stationary and suspension cultures

The growth characteristics of the BM-5 insect cell line of Bombyx mori (silkworm) have been experimentally investigated in order to develop optimal growth protocols when these cells are used to produce large quantities of biopesticides or human proteins by recombinant baculoviruses. Experiments were performed in 2 mL wells and 200 mL spinner flasks. Spinner flasks were operated at 80 rpm with 0.3% methyl cellulose (MCL) added to the medium in order to protect the cells from liquid shear stress. In addition to the effect of agitation rate and amount of MCL added to the medium, the cell response during the adaptation to growth in suspension from stationary cultures is reported. Exposure of the cells to varying nutrient and metabolite concentrations is accomplished through batch and repeated-batch modes in 2 mL wells. The results imply that glutamine is a limiting nutrient and lactate has an inhibitory effect on cell growth. Ammonia depletion from the medium was accompanied by uric acid accumulation, suggesting that ammonia is converted to this metabolic product by the “uricotelic” and “nucleicolytic” metabolic pathways.     

细胞固定化条件对紫草细胞生产紫草色素的影响

以海藻胶做包埋剂,研究了固化液构成、细胞包埋量和胞龄对紫草色素合成的影响。结果表明：固定化细胞合成紫草色素的合适固化液为含有０．１ｍｏｌ／ＬＣａＣｌ２的紫草色素生产培养基,该固化条件比较温和,并可长久保持固定化细胞活性;最适宜的细胞包埋质量分数为１０％２０％;用于细胞包埋的最佳细胞生长时间为１７ｄ。对紫草细胞固定化培养生产紫草色素过程的动力学特征进行了分析,建立了基质消耗和色素合成的动力学模型,并用该模型对实验数据进行了回归分析,实验数据与理论值之间具有比较令人满意的一致性     

Insect‐Specific Production of New GameXPeptides in Photorhabdus luminescens TTO1, Widespread Natural Products in Entomopathogenic Bacteria

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect‐specific production of the new GameXPeptides E–H ( 5 – 8 ) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions.     

Microbial Volatile Emissions as Insect Semiochemicals

We provide a synthesis of the literature describing biochemical interactions between microorganisms and insects by way of microbial volatile organic compound (MVOC) production. We evaluated the functionality and ecological context of MVOC signals, and explored important metabolic pathways involved in MVOC production. The cosmopolitan distribution of microorganisms creates a context for frequent, and frequently overlooked, insect responses to microbial emissions. There are numerous instances of MVOCs being closely associated with insect feeding behaviors, but some MVOCs are also powerful repellants. Emissions from microorganisms in situ may signal aspects of habitat suitability or potential exposure to entomopathogens. In some ecosystems, bacterial or fungal volatiles can also incite insect aggregations, or MVOCs can resemble sexual pheromones that elicit mating and oviposition behaviors from responding insects. A single microorganism or MVOC can have different effects on insect behaviors, especially across species, ontogenies, and habitats. There appears to be a multipartite basis for insect responses to MVOCs, and complex tritrophic interactions can result from the production of MVOCs. Many biochemical pathways for behaviorally active volatile production by microbial species are conserved across large taxonomic groupings of microorganisms. In addition, there is substantial functional redundancy in MVOCs: fungal tissues commonly produce polyketides and short-chain alcohols, whereas bacterial tissues tend to be more commonly associated with amines and pyrazines. We hypothesize that insect olfactory responses to emissions from microorganisms inhabiting their sensory environment are much more common than currently recognized, and that these signals represent evolutionarily reliable infochemicals. Insect chemoreception of microbial volatiles may contribute to the formation of neutral, beneficial, or even harmful symbioses and provide considerable insight into the evolution of insect behavioral responses to volatile compounds.