食品级乳酸菌表达系统研究进展

乳酸菌表达系统是近几年发展起来的食品级高效表达系统。乳酸菌具有益生菌特征,因此该表达系统与其他细菌表达系统相比有很多优点。介绍了糖诱导表达系统、噬菌体Φ31爆发式诱导的表达系统、乳链球菌素调控表达系统、温控表达系统等的研究进展,以及这些系统的应用前景。     

巴斯德毕赤酵母表达外源蛋白的研究进展

毕赤酵母表达系统是近年来发展起来的一种高效表达外源基因的表达系统。综述了毕赤酵母表达系统的起源、生物学特性、融合蛋白的表达以及影响蛋白表达量的因素。     

重组蛋白质在大肠杆菌体系中的可溶性表达策略

大肠杆菌表达体系因其表达量高、周期短、成本低等诸多优势特征而被广泛用作重组异源蛋白质的表达宿主。据统计超过30%的重组蛋白质药物和50%重组蛋白质的制备是使用大肠杆菌作为表达宿主。蛋白质错误折叠或未折叠以及包涵体形成是大肠杆菌表达体系更广泛应用的主要阻碍。因此,重组蛋白质在大肠杆菌体系中可溶性表达策略探索意义重大。综述了重组蛋白质在大肠杆菌表达系统中不可溶性表达的原因、机制以及影响大肠杆菌表达系统重组蛋白质可溶性的一些关键因素,并基于外源蛋白质在大肠杆菌中表达的各个步骤,总结了目前促进蛋白质在大肠杆菌表达系统中高效、可溶性表达的策略,为进一步拓展大肠杆菌表达体系在重组异源蛋白质可溶性表达中的应用提供参考。     

新型大肠杆菌高效表达载体pHsh的构建与应用

在现代生物学和生物技术研究中,通过基因的重组表达获得目标蛋白是一种应用最广泛的方法。因其培养简单、操作方便、遗传背景清楚、克隆表达系统成熟完善,大肠杆菌表达系统通常是人们表达重组蛋白的首选,而表达载体在重组蛋白的生产中起决定作用。pHsh及其衍生质粒是近年发展起来的新型大肠杆菌表达载体,其调控外源基因表达的原理不同于所有其他表达系统,并且具有表达水平高、成本低廉等特点。介绍大肠杆菌表达系统的组成和常用表达载体,并对由pHsh系列载体组成的Hsh表达体系的构建策略、表达调控机制及其使用方法进行综述。Hsh表达体系的建立和发展有望从一个不同的角度帮助解决基因的重组表达中常见的表达水平低、诱导剂成本高、包涵体形成等问题。     

2个表达单元间相互位置和转录方向对表达的影响

目的:研究质粒表达载体中2个表达单元间相互位置和转录方向关系对表达的影响,找出利于2个表达单元表达的优化的相互关系。方法:以全抗体为研究对象,构建了轻重链方向不同的2种相互关系的单质粒表达载体pIRESdhfrA和pIRESdhfrB,转染CHO-dhfr-细胞后,对瞬时表达水平进行了比较。以pIRESdhfrA和pIRESdhfrB为基础,构建了瞬时表达载体pIRESdhfrA-sv40ori和pIRESdhfrB-sv40ori,在COS-7细胞中进行了瞬时表达水平的比较。构建了基于CHO定点细胞系的稳定表达细胞株,对pIRESdhfrA和pIRESdhfrB进行稳定表达水平的比较。结果:在CHO-dhfr-的瞬时表达水平比较中,pIRESdhfrB转染的细胞表达水平是pIRESdhfrA转染细胞的2.18倍。与pIRESdhfrA-sv40ori在COS-7细胞的瞬时表达水平相比,pIRESdhfrB-sv40ori是其表达水平的2.3倍。pIRESdhfrB-FRT构建的CHO定点细胞平均表达水平是pIRESdhfrA-FRT构建的CHO定点细胞的11.6倍。结论:在构建的真核细胞质粒表达载体pIRESdhfr中,2个表达盒的启动子头-头转录方向关系比头-尾方向关系更利于重组蛋白的表达。     

Cx43基因在人类及小鼠胎心发育中的时空表达规律

目的　检测Cx4 3在人类和小鼠的胚胎心脏的表达 ,了解该基因在心脏发育过程中的表达规律。方法　选取人类 6～ 18孕周正常胚胎或胎儿心脏 6 3例 ,小鼠孕龄 9 5～ 16 5d胚胎心脏 6 4例 ,采用免疫组化法显示Cx4 3基因在心脏的表达。结果　早期人类胚胎心脏中 ,Cx4 3在心室肌中没有表达 ,心房肌表达微弱 ,原始小梁网中表达很高 ,随着胚胎发育 ,在心房和心室的表达逐渐增强 ,小梁网的表达在胚胎 13～ 14周达到高峰。室间隔的肌部表达量较弱 ,膜部室间隔不表达。房室瓣和大动脉根部管壁Cx4 3没有明显表达。除了在大动脉管壁表达不同 ,小鼠胚胎心脏表达规律与人类基本相同。结论　Cx4 3对于胚胎心脏的发育至关重要。     

毕赤酵母表达系统

选择合适的表达系统是蛋白质体外成功表达的关键。毕赤酵母表达系统是一种新的、极具潜力的真核表达系统,其在蛋白质表达方面具有其它系统不可比拟的优点,正在得到越来越广泛的应用。较详细地综迷了毕赤酵母表达系统的特点、组成及影响其表达的因素等。     

抗IV型胶原酶单链抗体在毕赤酵母中分泌表达*

利用毕赤酵母系统表达抗 IV型胶原酶人单链抗体。首先把目的基因克隆到毕赤酵母表达载体上 ,电击转化受体菌。在甲醇诱导下表达单链抗体。 SDS- PAGE和免疫印迹显示毕赤酵母分泌表达人单链抗体 ,表达量约 2 0 mg/ L酵母培养物。该表达系统与大肠杆菌相比 ,简化了表达产物的分离纯化程序。     

恶性胸腔积液中肺腺癌细胞的EGFR、ERCC1、TYMS、TUBB3和RRM1的表达及分子生物学意义

目的探讨EGFR、ERCCl、TYMS、TUBB3和RRMl在肺腺癌中的表达及分子生物学意义,为晚期肺癌患者的联合化疗提供参考。方法选取恶性胸腔积液中肺腺癌患者112例,采用免疫细胞化学方法对其进行EGFR、ERCCl、TYMS、RRMl、TUBB3的表达检测并根据EGFR不同的表达强度来统计ERCCl、TYMS、TUBB3和RRMl表达率的差异。结果在112例恶性胸腔积液中EGFR的阴性表达率为0．9％,弱阳性表达率为15．2％,阳性表达率为40．2％,强阳性表达率为43．7％,ERCCl的阳性表达率为40．2％,TYMS的阳性表达率为41．1％,TUBB3的阳性表达率为75％,RRMl的阳性表达率为83％。在不同EGFR表达强度下,ERCCl、TYMS和TUBB3各蛋白表达率不同且具有差异性。结论根据上述蛋白表达情况的不同,可对临床选择不同化疗方案提供参考意见。     

Parkin基因过表达质粒和细胞模型的构建

目的:构建Parkin基因过表达质粒并转染SH-SY5Y细胞,为进一步研究帕金森病的发病机制及中药的作用环节奠定基础。方法:首先构建Parkin基因过表达质粒,采用脂质体转导技术,将Parkin过表达质粒应用Lipo3000转染SH-SY5Y细胞。荧光显微镜观察细胞绿色荧光蛋白的表达;RT-PCR检测其Parkin mRNA的表达;Western Blot技术检测其Parkin蛋白的表达。结果:转染组可观测到较多的绿色荧光蛋白表达;Parkin过表达细胞Parkin mRNA和蛋白的表达均显著提高(P0.01)。结论:通过脂质体转导技术,应用Lipo3000可将Parkin过表达质粒成功转染入SH-SY5Y细胞,转染的基因和蛋白表达均较高,提示此法可成功构建Parkin基因过表达的细胞模型。     

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.     

Phylogenetic revision of the Taenogera Kröber genus-group (Diptera: Therevidae), with descriptions of two new genera

The phylogenetic relationships of the Taenogera genus-group, consisting of nine genera, are examined using cladistic methods. Twenty-one species representing nine genera and one outgroup were compared in a cladistic analysis across 99 states in 44 characters. The genus Taenogera Kröber is revised to contain only T. longa (Schiner), T. nitida (Macquart) and T. notatithorax Mann. Two new genera are described and figured: Actenomeros gen. n., with two species, A. corniculaticaudus sp. n. and A. onyx sp. n.; and Taenogerella gen. n., with four species, Ta. elizabethae sp. n., Ta. platina sp. n., Ta. schlingeri sp. n. and Ta . nigrapicalis (Mann) comb. n. Phylogenetic analysis also supports the removal of Nanexila gracilis (Mann) comb. n. from Taenogera .     

Relationships between host blood factors and proteases in Glossina morsitans subspecies infected with Trypanosoma congolense

Abstract. Host blood effects on Trypanosoma congolense establishment in Glossina morsitans morsitans and Glossina morsitans centralis were investigated using goat, rabbit, cow and rhinoceros blood. Meals containing goat erythrocytes facilitated infection in G. m. morsitans , whereas meals containing goat plasma facilitated infection in G. m. centralis. Goat blood effects were not observed in the presence of complementary rabbit blood components. N-acetyl-glucosamine (a midguMectin inhibitor) increased infection rates in some, but not all, blood manipulations. Cholesterol increased infection rates in G. m. centralis only. Both compounds together added to cow blood produced superinfection in G. m. centralis , but not in G. m. morsitans. Midgut protease levels did not differ 6 days post-infection in flies maintaining infections versus flies clearing infections. Protease levels were weakly correlated with patterns of infection, but only in G. m. morsitans. These results suggest that physiological mechanisms responsible for variation in infection rates are only superficially similar in these closely-related tsetse.     

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Evaluation of the Palaearctic Eurithia consobrina (Diptera: Tachinidae) as a Potential Biocontrol Agent for Mamestra configurata (Lepidoptera: Noctuidae) in Canada

The evaluation of Eurithia consobrina (Meigen) as a candidate for introduction against bertha armyworm, Mamestra configurata (Walker) (Lepidoptera: Noctuidae), in western Canada included studies of its host range, distribution, synchronization with host, constancy, abundance and life history in Europe. In addition, its diapause induction and its coldhardiness were compared with those of the target host M. configurata and its native tachinid parasitoid, Athrycia cinerea (Coq.). E. consobrina was found to fit criteria for a successful biocontrol agent reasonably well. It also had the potential to fill a largely unoccupied niche in the parasitoid complex of M. configurata. E. consobrina has a facultative diapause of the long-day type, similar to A. cinerea, but is less sensitive to diapause-inducing conditions. E. consobrina is at least as coldhardy as A. cinerea and more coldhardy than M. configurata. Laboratory exposures to temperatures of 0 C for 140 days apparently selected for greater coldhardiness, and later generations survived exposures of 140 days at -15 C without significant mortality. Adult E. consobrina were released at three locations in Manitoba in 1986 and 1987, but establishment has not been confirmed.     

Predation by Halmus chalybeus (Coleoptera: Coccinellidae) on Ceroplastes destructor and C. sinensis (Hemiptera: Coccidae: Ceroplastinae) Infesting Citrus in Northland, New Zealand

Predation by Halmus chalybeus (steelblue ladybird) on two species of wax scale was studied on citrus orchards in Northland, New Zealand. Field experiments using an exclusion technique of enclosing citrus branches in bags, found that larval and adult H. chalybeus preyed on first and second instars of both Ceroplastes destructor (white wax scale) and C. sinensis (Chinese wax scale), but not third instar C. destructor. Third instar C. sinensis and adults of both species were not tested but are rarely, if ever, attacked by H. chalybeus. The sampling of scale and ladybird populations and field experiments showed that few scales of either species survived past the second instar stage where H. chalybeus was numerous. The feeding rate of H. chalybeus on settled first and second instar scales was estimated inside bagged branches. Adults consumed on average 15.6 C. destructor and 13.3 C. sinensis per day per ladybird, while larvae ate 9.7 C. destructor per day. These feeding rates accounted for the experimental reduction of scale populations. H. chalybeus is a useful natural enemy for the control of C. destructor and C. sinensis when its activity is not disrupted by pesticides.     

Further flavonoid studies onAttalea species and some related cocosoid palms

In a flavonoid survey of direct and hydrolysed leaf extracts of sixteenAttalea, sevenScheelea and fourOrbignya species free tricin, tricin 7-glycosides, tricin 5-glucoside and flavone C-glycosides were the most frequent constituents; present in 100, 89, 70, and 81% of species, respectively. Luteolin, quercetin and isorhamnetin were each found in only 15% of the sample. The present results confirm the findings of a previous survey thatAttalea, Scheelea andOrbignya are chemically heterogeneous with as much variation between species as between genera. Furthermore, threeAttalea species,A. allenii, A. guaranitica andA. victoriana showed some infraspecific variation. On the other hand all three accessions ofA. ferruginea and six ofA. geraensis examined gave identical flavonoid profiles. The results support the view thatA. geraensis andA. guaranitica are closely related but do not support the suggested close relationship based on morphology betweenA. oleifera, A. burretiana andA. piassabossu.     

Investigations into the evolutionary history of the polyploid complexAntennaria neodioica (Asteraceae: Inuleae)

TheAntennaria neodioica polyploid agamic complex is a polymorphic species occurring across North America mainly north of the terminal margin of the Wisconsin glacier. This taxonomically difficult group has recently been treated as consisting of the four subspeciesA. neodioica subsp.canadensis, subsp.howellii, subsp.neodioica, and subsp.petaloidea. TheA. neodioica agamic complex has been considered of hybrid origin with several sexual diploid species constituting its parentage. Crosses were made among five sexual diploid species ofAntennaria, morphologically similar toA. neodioica s.l., in an attempt to discover its origins. Representative specimens of the five diploid species,A. neodioica s. l., and the synthetic interspecific F₁ hybrids were subjected to various analyses including PCA, cluster (UPGMA), and discriminant analyses. Results suggest that theA. neodioica complex is of multiple hybrid origin involving the four diploid speciesA. neglecta, A. plantaginifolia, A. racemosa, andA. virginica. BecauseA. neodioica is the result of diverse origin it is more desirable to consider the agamic complex as a separate, distinct species from its sexual diploid relatives. Several morphological characters in the diploid species were determined to be polygenically inherited.Investigations into the Evolutionary History of the Polyploid Complexes inAntennaria (Asteraceae: Inuleae). I.