提高哺乳动物工程细胞抗凋亡能力的基因策略

利用哺乳动物细胞发酵生产重组蛋白药物具有细菌、酵母等表达系统所不具备的显著优势,因此在生物制药工程中的重要性越来越突出。哺乳动物细胞对工业生产环境下的各种应激环境耐受能力差,易发生细胞凋亡,严重阻碍了大规模生产,降低了生产效率。细胞凋亡是细胞必经的生物学过程,随着对凋亡机制的深入了解,发展出各种抗凋亡策略,并有望应用于重构更适合工业生产的工程细胞。常用的抗凋亡策略包括:下调凋亡蛋白、上调抗凋亡蛋白、增强生长因子自表达、减少有毒代谢产物生成等。以上策略虽然能在一定程度上提高细胞的抗凋亡能力,但距离满足生产的工程细胞重构还有距离,围绕提高工程细胞的抗凋亡能力,已发展出"凋亡工程"这一重要的技术领域。     

用于生产重组蛋白药物的抗凋亡CHO宿主细胞株的建立

哺乳动物工程细胞在大规模培养生产重组蛋白时很容易发生细胞凋亡,从而导致生产过程提前终止,造成生产成本高昂。细胞代谢产物氨已被证明可以促进细胞凋亡,而线粒体膜整合蛋白Bcl-2可以通过促进线粒体膜完整性而抑制细胞凋亡。本实验应用谷氨酰胺合成酶加压系统在CHO工程细胞中高效表达中国仓鼠Bcl-2蛋白,使细胞具有抗凋亡能力的同时,利用谷氨酸和氨合成谷氨酰胺而有效降低培养基中氨的含量,从而达到抑制细胞凋亡的目的。     

基因编辑在构建抗凋亡工程细胞中的应用

21世纪以来,人们在生物科技领域取得了长足进步。一方面,基因编辑技术的问世为精确修饰目的基因带来可能,即能够在活细胞中完成特定DNA片段的插入、删除、替换、激活、抑制等任务;另一方面,越来越多的新型重组蛋白药物被开发并应用于对抗肿瘤等人类重大疾病,相比于传统化学药物,它具有高特异性和低副作用等显著优势,治疗效果得到普遍认可。目前用于生产治疗性重组蛋白的工程细胞株主要来源于哺乳动物,然而,由于生产过程中任何环境因素的改变都可能使工程细胞凋亡,严重影响蛋白的表达水平,因此大大提高了生产成本。研究人员在明确工程细胞的生长与死亡相关机制后,利用基因编辑技术对其进行了定向改造,提高了其表达水平。该文就对该方面研究成果进行了综述。     

用于重组蛋白生产的哺乳动物细胞表达新技术

近年来,用于重组蛋白生产的哺乳动物细胞表达领域涌现出一系列革命性的新技术。优化的工程细胞为表达重组蛋白提供了优良的宿主;基于荧光的筛选方法可以快捷地得到高表达细胞株;高通量的培养工艺能够预测适合外源蛋白表达的细胞培养条件;可抛弃式生物反应器为大规模细胞培养提供了更多的选择;大规模瞬时表达技术节省了重组蛋白的生产时间。这些新技术提高了重组蛋白的研发和生产效率,加快了蛋白药物的工业化进程。     

乳腺生物反应器的研究现状

乳腺生物反应器是将外源基因在哺乳动物的乳腺中特异表达,以转基因动物的乳腺组织生产药用蛋白。采用乳腺生物反尖器生产药用蛋白质是一种全新的生产模式,它已经成为生物技术领域发展的重要方面。乳腺生物反应器具有能够生产出具有完全生物活性的药用收白,纯化简单,投资少,成本低,对环境没有任何污染等优点,转基因动物生产药用蛋白可以获得巨额经济效益。     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

利用转基因植物生产生物药[英国]威尔士大学生物科学系Glynis Giddings等原著

传统的生物技术常利用哺乳动物细胞、细菌和真菌培养作为转基因系统,来生产生物药.鉴于今后对生物药例如治疗贫血的红细胞生成素和治疗糖尿病的胰岛素,以及通过基因组研究发现的新的药用蛋白的需求量均将大量增加,故有必要利用另一种转基因系统来生产价格既低廉,使用又安全的重组生物药.     

糖蛋白药物表达系统糖基化研究进展

随着基因重组技术的不断发展和蛋白糖基化机制研究的不断深入,糖蛋白药物对人类健康的重要作用越来越受到重视.迄今为止,哺乳动物表达载体仍然是最常见的药用蛋白生产系统.由于其存在成本高、产率低等问题,酵母和细菌表达系统也日益受到重视.受到理论和技术的限制,细菌表达系统的开发仍存在相当大的困难,而酵母表达系统的开发和应用已经取得了一系列突破性的成果.本文综述了国内外近年来改造不同表达系统N-糖基化途径用于生产人源糖蛋白的研究进展.     

利用转基因植物生产生物药

传统的生物技术常利用哺乳动物细胞、细菌和真菌培养作为转基因系统 ,来生产生物药。鉴于今后对生物药例如治疗贫血的红细胞生成素和治疗糖尿病的胰岛素 ,以及通过基因组研究发现的新的药用蛋白的需求量均将大量增加 ,故有必要利用另一种转基因系统来生产价格既低廉 ,使用又安全的重组生物药。认为适合于上述目的者为高等植物。利用转基因植物来生产重组药用蛋白和肽有很多优点 ,例如 :(1)植物容易转化 ;(2 )使用安全 ,因植物不是人类病原体的宿主 ,故与动物细胞培养比较 ,利用重组植物生产的生物药不论提纯与否 ,都不大有可能污染人类病原…     

哺乳动物细胞表达系统研究进展

目前,哺乳动物细胞已成为生产多种生物药物的首选宿主细胞。哺乳动物细胞表达系统可进行翻译后修饰,表达的重组蛋白接近人源构象,因此被大量用于治疗性重组蛋白的生产,如何建立高效的哺乳动物细胞表达系统也受到研究者们的重视。随着基因组学、转录组学、蛋白质组学以及代谢组学研究不断深入,近几年来在优化哺乳动物表达系统方面取得了长足的进步。从高效表达载体构建、宿主细胞改造、高通量筛选、培养基优化等方面阐述哺乳动物细胞表达系统的研究进展,以期为研究者们提供一定的帮助。     

Materials for Chinese Phyllanthoideae

The present paper is part of taxonomic study on Chinese Phyllanthoideae. Included in it are two new varieties, Leptopus esquirolii var. villosus and Drypetes hainanensis var. longistipitata, one new combination, Glochidion triandrum var. siamense, and seven new records in China: Drypetes salicifolia, D. hoaensis. Actephila subsessilis, Glochidion khasicum, G. nubigennum, Bridelia spiosa and B. poilanei. In addition, seventeen taxon names are newly reduced: Liodendron formosanum = Drypetes formosana, Liodendron matsumurae = Drypetes matsumurae, D. longipes = D. indica, Antidesma paxii = A. acidum, A. hiiranense, A. filipes and A. pentandrum var. hiiranense = A. japonicum, A. calvescens = A. montanum, A. microphyllum = A. venosum, Breynia stipitata var. formosana and B. jormosana = B. vitis-idaea, Glochidion zeylanicum var. tomentosum = G. hirsutum, G. rubidulum = G. thomsonii, G. acuminatum = G. triandrum, G. fagifolium and Phyllanthus fagifolius = Glochidion sphaerogynum, Bridelia penangiena = B. insulana, B. henryana = B. tomentosa. All the types are kept in SCBl and PE.     

Neutrino intergalactic communication,metal life,and viruses: Part 1 quo vadis ex machina

At one spectrum extreme, Astrobiology conjectures that for exoplanets with Goldilocks conditions, terrestrial-like life is inevitable. Moreover, it is envisaged that via panspermia, terrestrial-like life and its precursors are transferred among galaxies, stars, and within solar systems via transiting comets, asteroids, and planetoids. In addition, expelled stars, which have solar systems, it is inferred, transfer life as well. However, at the other extreme, we propose a paradigm shift that on some planets, subject to non- Goldilocks conditions, metal machine life could arise, ab initio, and evolve viruses, intelligence, and civilizations, conjointly. Accordingly, intelligent mechanized civilizations could readily and efficiently commence space exploration. Furthermore, as a counter paradigm shift, such civilizations could experiment and produce non-metallic life, based on carbon and other non-metal elements, under suitable conditions, related to Goldilocks life. Even a single example of validated interstellar or intergalactic communication received on the Earth would support the existence of life elsewhere. However, the communication platform should not be restricted to electromagnetic radiation. Other platforms should be included as well - one such example, which would require sophisticated technology, is neutrino communication. This is the case for any advanced civilization, be it metal-machine based, biological-based, and carbon-based. In sum, civilizations based on machine life, would be highly productive due to the longevity and hardiness of machine life. However, significant caveats are raised in this brief report, because possibly dissimilar psychologies and intelligence may lead to conflicts between metal machine life and biological life, inter-paradigm conflict.     

Nicotinic Agonists, Antagonists, and Modulators From Natural Sources

1. Acetylcholine receptors were initially defined as nicotinic or muscarinic, based on selective activation by two natural products, nicotine and muscarine. Several further nicotinic agonists have been discovered from natural sources, including cytisine, anatoxin, ferruginine, anabaseine, epibatidine, and epiquinamide. These have provided lead structures for the design of a wide range of synthetic agents.2. Natural sources have also provided competitive nicotinic antagonists, such as the Erythrina alkaloids, the tubocurarines, and methyllycaconitine. Noncompetitive antagonists, such as the histrionicotoxins, various izidines, decahydroquinolines, spiropyrrolizidine oximes, pseudophrynamines, ibogaine, strychnine, cocaine, and sparteine have come from natural sources. Finally, galanthamine, codeine, and ivermectin represent positive modulators of nicotinic function, derived from natural sources.3. Clearly, research on acetylcholine receptors and functions has been dependent on key natural products and the synthetic agents that they inspired.     

Molecular phylogeny of the gobioid fishes (Teleostei: Perciformes: Gobioidei)

The phylogeny of groups within Gobioidei is examined with molecular sequence data. Gobioidei is a speciose, morphologically diverse group of teleost fishes, most of which are small, benthic, and marine. Efforts to hypothesize relationships among the gobioid groups have been hampered by the prevalence of reductive evolution among goby species; such reduction can make identification of informative morphological characters particularly difficult. Gobies have been variously grouped into two to nine families, several with included subfamilies, but most existing taxonomies are not phylogenetic and few cladistic hypotheses of relationships among goby groups have been advanced. In this study, representatives of eight of the nine gobioid familes (Eleotridae, Odontobutidae, Xenisthmidae, Gobiidae, Kraemeriidae, Schindleriidae, Microdesmidae, and Ptereleotridae), selected to sample broadly from the range of goby diversity, were examined. Complete sequence from the mitochondrial ND1, ND2, and COI genes (3573 bp) was used in a cladistic parsimony analysis to hypothesize relationships among the gobioid groups. A single most parsimonious topology was obtained, with decay indices indicating strong support for most nodes. Major phylogenetic conclusions include that Xenisthmidae is part of Eleotridae, and Eleotridae is paraphyletic with respect to a clade composed of Gobiidae, Microdesmidae, Ptereleotridae, Kraemeriidae, and Schindleriidae. Within this five-family clade, two clades are recovered. One includes Gobionellinae, which is paraphyletic with respect to Kraemeriidae, Sicydiinae, Oxudercinae, and Amblyopinae. The other contains Gobiinae, also paraphyletic, and including Microdesmidae, Ptereleotridae, and Schindleriidae. Previous morphological evidence for goby groupings is discussed; the phylogenetic hypothesis indicates that the morphological reduction observed in many goby species has been derived several times independently.     

Systematics and phylogeny of the caryophyllidia-bearing dorids (Mollusca, Nudibranchia), with descriptions of a new genus and four new species from Indo-Pacific deep waters

The phylogenetic relationships of the caryophyllidia-bearing dorids are studied, based on the examination of the type species of all the genera previously described. The phylogenetic hypothesis supports that the caryophyllidia-bearing dorids are a monophyletic group and the sister group of the clade formed by Astemnotus Ehrenberg, 1831 and Halgerda Bergh, 1880. Several genera previously considered as valid or regarded as uncertain are here synonymized: Peronodoris Bergh, 1904, Trippa Bergh, 1877, Phlegmodoris Bergh, 1878, Petelodoris Bergh, 1881, Kentrodoris Bergh, 1876, Audura Bergh, 1878, Centrodoris P. Fischer, 1883, Anisodoris Bergh, 1898, Awuka Er. Marcus, 1955, Rhabdochiia P. Fischer, 1883, Boreodoris Odhner, 1939, Dictyodoris Bergh, 1880, Gravieria Vayssiere, 1912, Aporodoris Ihering, 1886. The following genera are regarded as valid: Astemnotus, Atagema J.E. Gray, 1850, Jorunna Bergh, 1876, Platydoris Bergh, 1877, Diaulula Bergh, 1878, Rostanga Bergh, 1879, Halgerda Bergh, 1880, Baptodoris Bergh, 1884, Gargamella Bergh, 1894, Alloiodoris Bergh, 1904, Sclerodoris Eliot, 1904, Taringa Er. Marcus, 1955, Thorybopus Bouchet, 1977. The new genus Nophodoris is described based on two new species from New Caledonia deep waters. Two additional new species from New Caledonia belonging to the genera Atagema and Gargamella are also described. Nomenclatural and taxonomic problems are discussed, and several type species, neotypes and lectotypes are selected.     

Revision of the Malagasy genus Trichoteleia Kieffer (Hymenoptera, Platygastroidea, Platygastridae)

The species of the genus Trichoteleia Kieffer (Hymenoptera: Platygastridae) are revised: 42 species are recognized, of which two were previously named and are redescribed: Trichoteleia afo Talamas, sp. n., Trichoteleia albidipes Kieffer, Trichoteleia bicolor Talamas, sp. n.; Trichoteleia bidentata Talamas sp. n.; Trichoteleia carinata Talamas, sp. n.; Trichoteleia cincta Talamas & Masner, sp. n.; Trichoteleia delilah Talamas, sp. n.; Trichoteleia eburata Talamas, sp. n.; Trichoteleia echinata Talamas, sp. n.; Trichoteleia fisheri Talamas & Masner, sp. n.; Trichoteleia funesta Talamas, sp. n.; Trichoteleia halterata Talamas & Masner, sp. n.; Trichoteleia hemlyae Talamas & Masner, sp. n.; Trichoteleia irwini Talamas & Masner, sp. n.; Trichoteleia janus Talamas, sp. n.; Trichoteleia jiro Talamas, sp. n.; T. ketrona Talamas, sp. n.; Trichoteleia levii Talamas & Johnson, sp. n.; Trichoteleia longiventris Talamas & Masner, sp. n.; Trichoteleia minima Talamas, sp. n.; Trichoteleia nify Talamas & Masner, sp. n.; Trichoteleia oculea Talamas, sp. n.; Trichoteleia orona Talamas & Masner, sp. n.; Trichoteleia parvipennis Talamas & Masner, sp. n.; Trichoteleia pauliani (Risbec); Trichoteleia picturata Talamas, sp. n.; Trichoteleia prima Talamas, sp. n.; Trichoteleia prolixa Talamas, sp. n.; Trichoteleia quazii Talamas, sp. n.; Trichoteleia ravaka Talamas, sp. n.; Trichoteleia rugifrons Talamas & Masner, sp. n.; Trichoteleia solocis Talamas, sp. n.; Trichoteleia sphaerica Talamas, sp. n.; Trichoteleia subtilis Talamas & Masner, sp. n.; Trichoteleia tahotra Talamas & Masner, sp. n.; Trichoteleia takariva Talamas, sp. n.; Trichoteleia tezitra Talamas, sp. n.; Trichoteleia tigris Talamas, sp. n.; Trichoteleia tonsa Talamas, sp. n.; Trichoteleia warreni Talamas & Masner, sp. n.; Trichoteleia xantrox Talamas, sp. n.; Trichoteleia zuparkoi Talamas & Masner, sp. n. A neotype is designated for Trichoteleia albidipes and a lectotype is designated for Trichoteleia pauliani.     

Oxypred： Prediction and Classification of Oxygen-Binding Proteins

This study describes a method for predicting and classifying oxygen-binding pro- teins. Firstly, support vector machine (SVM) modules were developed using amino acid composition and dipeptide composition for predicting oxygen-binding pro- teins, and achieved maximum accuracy of 85.5% and 87.8%, respectively. Sec- ondly, an SVM module was developed based on amino acid composition, classify- ing the predicted oxygen-binding proteins into six classes with accuracy of 95.8%, 97.5%, 97.5%, 96.9%, 99.4%, and 96.0% for erythrocruorin, hemerythrin, hemo- cyanin, hemoglobin, leghemoglobin, and myoglobin proteins, respectively. Finally, an SVM module was developed using dipeptide composition for classifying the oxygen-binding proteins, and achieved maximum accuracy of 96.1%, 98.7%, 98.7%, 85.6%, 99.6%, and 93.3% for the above six classes, respectively. All modules were trained and tested by five-fold cross validation. Based on the above approach, a web server Oxypred was developed for predicting and classifying oxygen-binding proteins(available from http://www.imtech.res.in/raghava/oxypred/).     

Mating systems of some putative polypore — agaric relatives

Mating systems are reported for taxa of polypores and agarics considered related. Taxa areDictyopanus pusillus, Favolus alveolaris, F. tenuiculus, Lentinus bertierii, L. crinitis, L. punctaticeps, L. strigellus, L. strigosus, L. suavissimus, Pleurotus australis, P. levis, P. tuberregium, Polyporus ciliatus, P. elegans, P. squamosus, andP. varius. All are tetrapolar exceptP. elegans.     

Some New Taxa and Records of Characeae in Hunan,China

In this paper, one new species, four new varieties and thirteen new records of Characeae from Hunan, China, are reported. They are Chara quadriscutulum, sp. nov., Nitella axilliformis var. biformis, var. nov., N. microcarpa var. unicarpa, var. nov., Chara braunii var. cylindrospora, var. nov., C. hydropitys var. hunanensis, var. nov., Nitella microcarpa Braun, N. leptoclada Braun, N. annandalei Pal, N. confervacea A. Braun, N. gracilliformis J. Groves, N. inversa Imahori, N. musashiensis Morioka, N. paucicostata T. F. Allen, N. acuminata var. gollmeriana (A. Br.) Zanev. et Wood, Tolypella boldii Sawa. Chara longifolia Robinson, C. pistianensis J. Vilhelm, C. grovesii Pal.