家蚕生物反应器的研究概述

家蚕生物反应器在生产活性成分、表达外源基因、改善蚕丝性能等方面,具有很高的开发利用价值。本文概述了家蚕幼虫反应器、家蚕NPV表达系统反应器、转基因家蚕反应器、家蚕丝腺反应器的研究状况。     

以蚕蛹生产生物活性物质的操作方法

近年来,随着基因工程和重组技术的发展,家蚕作为生物反应器(家蚕杆状病毒表达载体系统)生产重组生物活性物质的优良特性被生物学家所认识,许多重组生物活性物质通过家蚕这一优良生物反应器得到表达和生产。如中国农业科学院蚕业研究所农业部家蚕生物技术重点实验室用家蚕高效表达了植酸酶基因.用于饲料添加剂,可大幅度减少饲料中无机磷的添加量.提高了料肉比,并减少了磷对环境的排放。又如浙江大学生物化学研究所用家蚕高效表达hGM-CFS制备口服药具有显著的升白细胞功能。     

家蚕性别与乙酰胆碱酯酶基因(ace1、ace2)表达的关系研究

为了研究家蚕性别与两种乙酰胆碱酯酶基因转录表达的关系,采用辛硫磷对5龄第3d的雌雄家蚕进行诱导,采用半定量RT-PCR方法,研究两基因在雌雄家蚕的脑和脂肪体中的表达特征。研究结果表明,农药诱导后在雌雄家蚕的脑表达量均有所增加,雌家蚕ace1,雄家蚕ace1,雌家蚕ace2,雄家蚕ace2表达程度分别增加了70.50%、5.76%、217.00%、35.83%;在家蚕的脂肪体中ace1的表达量减少,ace2的表达量略有增加,且雌家蚕ace1,雄家蚕的ace1表达程度分别减少了5.37%、34.90%,雌家蚕ace2雄家蚕ace2表达量分别增加了9.32%、6.96%。研究表明,ace1和ace2在雄家蚕的脑组织中的表达量高于雌性家蚕,推测这可能是导致雌雄家蚕对农药抗性差异的原因;在农药诱导后,ace2在脂肪体里的表达和ace1的表达趋势相反,推测ace2在农药中毒发挥更加重要的代谢作用。本研究结果为家蚕的抗性品种选育和开发针对鳞翅目害虫的高效杀虫剂提供了新的信息。     

家蚕浓核病毒的研究

家蚕浓核病毒是一种使蚕发生软化病的病原,主要感染家蚕的中肠圆筒型细胞。家蚕浓核病毒可作为基因转移、表达的载体和农林害虫的生物防治,同时也是家蚕的四大病毒性疾病之一,对蚕桑生产危害巨大。现就病理学特征、基因组结构、感受性、检测方法和浓核病的防治等方面对家蚕浓核病毒进行概述。     

家蚕微孢子虫伴侣蛋白CCTθ的鉴定

家蚕微粒子病是一种严重危害蚕种生产的蚕病,被列为蚕桑生产唯一的检疫对象.分子伴侣协助底物蛋白折叠、装配和转运,但目前对家蚕微孢子虫分子伴侣CCT(chaperonin containing tailless complex polypeptide)的研究较少.本研究克隆了家蚕微孢子虫NbCCTθ基因,构建原核表达载体pET-28a-NbCCTθ表达重组蛋白,纯化后制备多克隆抗体.间接免疫荧光分析结果显示,NbCCTθ在家蚕微孢子虫的整个生命周期中均表达,但在不同发育阶段分布不同;在成熟孢子中主要分布于细胞质和质膜上,在裂殖增殖期主要分布于细胞质中,孢子形成期后期定位在细胞核中.研究结果为进一步探索NbCCTθ的具体功能奠定了基础,对家蚕微粒子病的防治也有一定的指导意义.     

影响家蚕表达外源基因效率的几个重要因子的比较研究

本研究以重组病毒rSj14NPV为感染病毒,通过比较纯化蛋白的产量,对家蚕作为生物反应器生产基因工 程产品的适宜蚕品种与相关的重组病毒接种技术进行了比较研究。研究结果表明:871×872、秋风×白玉为表达 Sj14的适宜蚕品种;重组病毒接种量与蛋白表达量有关,适宜接种量为4～5μL/蚕;适宜接种时间以5龄2日龄为 优;以家蚕表达外源基因,最好将重组病毒以家蚕细胞复苏后再接种家蚕以保证获得较高的表达效果。     

家蚕Polh+ Bac-to-Bac杆状病毒表达系统的构建

为利用杆状病毒的强启动子——多角体启动子而构建的重组杆状病毒一般是多角体缺失型(polh-)病毒。为了解决家蚕生物反应器规模化生产中病毒必需经皮接种而效率低下的问题,在建立家蚕Bac-to-Bac快速表达系统的基础上,构建了能形成多角体的家蚕Polh+ Bac-to-Bac表达系统。通过该系统能够产生表达外源目的基因的重组病毒,获得的该重组病毒能在培养细胞内表达,也能通过经口添食感染表达。利用EGFP报告基因分析了该系统的表达效果,构建的重组病毒不仅有效表达了EGFP蛋白,还在细胞核中形成了大量多角体。该系统较好解决了重组病毒必需经皮接种感染的缺陷,提高了生产效率,拓宽了杆状病毒在生物杀虫剂、基因治疗等领域的应用前景。     

家蚕转基因技术的应用研究

家蚕(Bombyx mori)是重要的经济昆虫,也是生物学研究的模式生物之一,中国在2004年率先发布家蚕基因组框架图,为中国在家蚕分子生物学领域占领了一个制高点。继框架图之后,进行转基因家蚕研究具有重要的理论意义和实用价值,这不仅可以帮助我们了解家蚕基因的结构、功能及表达调控的机制,同时也为研究家蚕生物反应器生产目的蛋白,培育家蚕抗病性新品种,改善蚕丝质量等提供了新的技术途径。本文将重点从家蚕功能基因组的研究、家蚕丝腺生物反应器的应用研究及转基因技术培育家蚕新品种三个方面对家蚕转基因技术的应用进行介绍。随着家蚕转基因技术的深入研究,转基因家蚕技术及产物将会在未来有着更广阔的应用前景。     

人工饲料育和桑叶育家蚕消化酶基因表达差异研究

α-淀粉酶、麦芽糖酶、脂肪酶和类胰蛋白酶与家蚕中肠对营养物质的消化密切相关。运用荧光定量PCR技术,对人工饲料育与桑叶育家蚕中肠中这4种消化酶基因在4、5龄期的表达情况进行了测试。结果表明,人工饲料育与桑叶育家蚕中4种消化酶的表达均存在较大差异,特别是人工饲料育家蚕5龄期的脂肪酶和类胰蛋白酶基因的表达量极显著低于桑叶育家蚕中的表达量,这可能影响到家蚕对营养物质的消化吸收、生长发育和抗病性。     

科技

<正>家蚕生物反应器家族再添新成员近日,农业农村部向中国农业科学院生物技术研究所颁发了重组杆状病毒rBmNPV表达的鸡γ-干扰素和猫ω-干扰素两项生产应用安全证书,标志着利用家蚕生物反应器生产动物用基因工程制品范围得到进一步拓宽。生物所微生物蛋白质工程创新团队通过多年研究,成功实现了鸡γ-干扰素在家蚕生物反应器中的重组表达。该产品具有生物活性好、生产成本低(每粒蚕蛹可产千万IU的干扰素、每只鸡全龄应用的直接成本约1分钱)、可     

杆状病毒表达系统在兽用亚单位疫苗领域应用的研究进展

杆状病毒表达系统是以杆状病毒为蛋白质表达载体,昆虫细胞或幼虫为受体的真核表达系统。与原核细胞相比,昆虫细胞可对外源蛋白进行翻译后修饰,如多肽折叠、糖基化、酰基化、磷酸化、二硫键形成及蛋白质水解等。因杆状病毒对畜禽无致病性,这使得杆状病毒表达系统成为一种安全有效的兽用亚单位疫苗生产平台。作者对杆状病毒表达系统在兽用亚单位疫苗领域中的应用作了简要综述。     

营养物质对基因表达的调控作用

营养物质作为调节基因表达的外部因子对基因表达起着非常重要的作用。作者综述了营养物质对基因表达调控的作用机制、调控方式及途径,并列出了几种营养物质对基因表达的调节作用。     

民猪生殖器官PRLR基因表达发育变化

试验以民猪为试验材料,长白猪为对照组,分别在1、2.5、4、6、8月龄时进行卵巢、输卵管和子宫取样,对促乳素受体（PRLR）基因表达的发育性变化规律进行研究。结果表明,生殖器官中PRLR的表达量与生殖器官的解剖指标存在正相关。PRLR在1、2.5、4、6和8月龄的民猪和长白猪的卵巢、输卵管和子宫中均有表达。民猪和长白猪卵巢PRLR mRNA表达水平从1月龄至8月龄呈现逐渐上升趋势,在8月龄达到最高水平,民猪卵巢PRLRmRNA表达水平显著高于长白猪（P〈0.05）。民猪和长白猪输卵管PRLR mRNA表达量随月龄增加呈上升趋势,在8月龄达到最高水平,品种间差异不显著。民猪子宫PRLR mRNA表达量随月龄增加而升高,在6月龄达到最高水平,8月龄显著下降（P〈0.05）,长白猪子宫PRLR mRNA表达量随月龄增加而升高,在8月龄达到最高水平,品种间在6月龄差异显著（P〈0.05）。     

Expression of drug efflux transporters in poultry tissues

Multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 2 (MRP2) are two prominent members of the super-family of ATP-binding cassette (ABC) transporters that carry a wide range of substrates across biological membranes, using ATP as energy source. The level of expression of these efflux transporters in different tissues has hitherto been studied mainly in mammals, and only P-glycoprotein (P-gp), the product of the MDR1 gene, has been described in chickens as of yet. The aim of this study was to describe the levels of expression of MDR1 and MRP2 mRNAs in different tissues of chickens, as these transporters play an important role in the absorption, distribution and excretion of drugs and toxins.In the gastro-intestinal tract, the highest levels of MDR1 mRNA expression were found in the small intestines, followed by the colon, whereas lower levels were found in the crop, proventriculus and the caeca. High MDR1 expression was also measured in the excretory organs such as liver, kidney and lungs. In contrast to rodents and humans, relatively low levels were found in the adrenals and in the immature sex organs such as testicles and ovaries.MRP2 mRNA expression was high in the liver, kidneys, duodenum and the jejunum, but expression was low in the ileum as well as in the lungs. No MRP2 expression could be detected in the other organs tested. Comparing the findings in chickens with previously published data, in particular those from humans and rodents, an unexpected high degree of similarity in the expression pattern of MDR1 and MRP2 mRNAs was apparent.     

营养对基因表达的影响

营养是基因表达的重要调节物。营养对基因表达的调控作用及其机制已成为分子生物学的重要研究内容及营养学的新兴研究领域。本文在介绍基因表达过程及营养调控途径的基础上 ,综述了能量与蛋白质、氨基酸、碳水化合物、脂肪与脂肪酸、矿物元素及维生素对相关基因表达的调控作用及主要机制     

Macronutrient modulation of mRNA and microRNA function in animals: A review

Dietary macronutrients have been regarded as a basic source of energy and amino acids that are necessary for the maintenance of cellular homeostasis, metabolic programming as well as protein synthesis. Due to the emergence of “nutrigenomics”, a unique discipline that combines nutritional and omics technologies to study the impacts of nutrition on genomics, it is increasingly evident that macronutrients also have a significant role in the gene expression regulation. Gene expression is a complex phenomenon controlled by several signaling pathways and could be influenced by a wide variety of environmental and physiological factors. Dietary macronutrients are the most important environmental factor influencing the expression of both genes and microRNAs (miRNA). miRNA are tiny molecules of 18 to 22 nucleotides long that regulate the expression of genes. Therefore, dietary macronutrients can influence the expression of genes in both direct and indirect manners. Recent advancements in the state-of-the-art technologies regarding molecular genetics, such as next-generation sequencing, quantitative PCR array, and microarray, allowed us to investigate the occurrence of genome-wide changes in the expression of genes in relation to augmented or reduced dietary macronutrient intake. The purpose of this review is to accumulate the current knowledge focusing on macronutrient mediated changes in the gene function. This review will discuss the impact of altered dietary carbohydrate, protein, and fat intake on the expression of coding genes and their functions. In addition, it will also summarize the regulation of miRNA, both cellular and extracellular miRNA, expression modulated by dietary macronutrients.     

Effect of Exosomes Derived from PPRV Vaccine Strain N75-1 Infected Cells on the Expression of SLAM in Goats

Signaling lymphocyte activation molecule (SLAM) was identified as the primary receptor of Peste des petits ruminants virus (PPRV) on goat lymphocytes. This study aimed to explore the effects of exosomes derived from PPRV vaccine strain N75-1 infected cells on the expression of SLAM in goats. In this study, SLAM expression and cytokines expression levels of goat PBMCs (recipient cells) incubated with exosomes isolated from PPRV-infected goat PBMCs (Exo-PPRV) was analyzed by quantitative real-time PCR, flow cytometric assay and Western blot assay. The results showed that the recipient cells treated with Exo-PPRV had significantly increased SLAM mRNA and cell surface expression (P<0.05) as compared with Exo-Mock treated cells. Furthermore, the increased TNF-α, IL-10, and IFN-γ expression as well as decreased IFN-α expression (P<0.05) were also detected. Further study demonstrated that Exo-PPRV that contained high levels of PPRV H protein could transmit H protein to recipient cells. Moreover, the expression level of SLAM in the pcDNA3.1-H transfected cells was significantly higher than that in the pcDNA3.1 transfected cells and non-transfection group. Taken together, our study demonstrated that PPRV infected cells derived exosomes has positive effects on the SLAM expression in the recipient cells, and that the high level of PPRV H protein contained in PPRV-Exo is one of the key molecules for exosomes to regulate the expression of SLAM in the recipient cells.     

Usage of putative chicken U6 promoters for vector-based RNA interference

Gene silencing with short interfering RNA (siRNA) expression vectors is a powerful method for the analysis of gene functions. For the expression of siRNA in mammalian cells, mammalian U6 small nuclear RNA (snRNA) promoters are widely used. However, the mammalian U6 promoter might not function well in other species. In this study, we cloned four putative chicken U6 promoters by PCR and analyzed their functions. First, we screened the chicken genomic database using the human U6 snRNA gene and identified four candidate sequences. The sequences contained some control elements in their promoter regions, but as we could not rule out that they were pseudogenes, we amplified these sequences and used them as promoters for short hairpin RNA (shRNA) expression. Using the firefly luciferase (Luc) gene as a target, transient expression assays were performed with chicken ovary-derived cells. All four putative chicken U6 promoters exhibited suppressive activity toward Luc, and so could act as a promoter for expression of the snRNA gene in the chicken genome. The promoter activity was not as strong as that of a commercially available siRNA expression vector. This probably reflects artificial sequences between the promoters and synthetic DNA encoding shRNA.     

Heat Shock Protein 70 and Sex Steroid Receptors in the Follicular Structures of Induced Ovarian Cysts

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERα than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERα in the treated group. An increase in total ERα protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERβ in animals with COD, and the total protein expression of ERβ was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.     

小反刍兽疫病毒疫苗毒株N75-1感染细胞源外泌体对山羊SLAM表达的影响

淋巴细胞信号活化分子（SLAM）是小反刍兽疫病毒（PPRV）在淋巴细胞的主要受体。本研究旨在探索PPRV感染细胞源外泌体对山羊SLAM表达的影响。通过荧光定量PCR法、流式细胞术和Western blot技术检测PPRV疫苗毒株N75-1感染山羊外周血单核细胞（PBMCs）源外泌体（Exo-PPRV）共育对接纳细胞（正常山羊PBMCs）中SLAM表达水平和细胞因子分泌水平的影响。结果表明,与正常细胞分泌外泌体（Exo-Mock）共育组相比,Exo-PPRV共培养组细胞SLAM mRNA和细胞表面表达水平均显著上调（P<0.05）,TNF-α、IL-10和IFN-γ表达水平升高,而IFN-α水平降低（P<0.05）。进一步研究表明,Exo-PPRV中荷载高水平的PPRV H蛋白且可以将其传递至接纳细胞中,同时pcDNA3.1-H转染组中SLAM表达水平较pcDNA3.1对照转染组和未转染组明显升高。以上结果表明,PPRV感染PBMCs源外泌体对接纳细胞中SLAM表达水平具有显著正调控作用,外泌体中载荷高水平PPRV H蛋白是其调控接纳细胞SLAM表达的关键分子之一。