Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

转基因海带配子体的制备与高效增殖

海带具有很高的营养价值和经济社会价值。自20世纪90年代以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，根据海带自身特点，建立了海带遗传转化体系（海带孢子体表达系统），它的基本原理是利用基因枪法转化海带配子体，经孤雌或受精途径再生幼孢子体后，用氯霉素筛选幼孢子体获得转基因海带，然后进行海上安全栽培和转基因产品的检测与提取。目前该表达系统已成功实现报告基因（β-半乳糖苷酶基因，lacZ）和功能基因（乙肝表面抗原基因，HBsAg）的稳定表达。 由于海带孢子体表达系统需经孢子体再生和海上栽培等阶段，周期较长，而且转基因安全性问题也在一定程度上制约其研究与应用。因此，我们在海带孢子体表达系统的基础上又建立和优化了海带配子体表达系统，并成功实现了报告基因（绿色荧光蛋白基因，GFP）的瞬间表达和功能基因（瑞替普酶基因，rt-PA）的稳定表达。虽然海带配子体表达系统能避免转基因安全性问题，周期较短，但在表达量和生物量积累方面，与孢子体表达系统相比还有较大差距。 本文首先在海带配子体表达系统中成功实现了人酸性成纤维细胞生长因子基因（hafgf）和鲎素基因（tac）的稳定表达，制备了转基因海带配子体，然后将光生物反应器培养技术应用于转基因海带配子体的高效增殖，以期解决阻碍海带配子体表达系统发展的量的问题，并为转基因海带配子体的大规模培养提供试验依据和技术支持。 本文的研究结果为： 1、人酸性成纤维细胞生长因子基因和鲎素基因可以稳定整合到海带配子体基因组中，实现转基因产物的表达。 2、根据转基因海带配子体的生长特点，研制开发了一套培养体积为300 ml的鼓泡式光生物反应器，它具有操作简便、成本低廉、适合海带配子体生长等特点。随后将培养体系扩大到2.5 L，并研究了光对转基因海带配子体生长的影响，试验结果显示，转基因海带配子体在光强为30 μE m-2 s-1时即可达到光饱和生长，最优光周期为14:10 LD，而且蓝光可促进转基因海带配子体的生长。 3、在前期研究工作的基础上，为改善反应器内的传质条件，我们又设计研制了2.5 L气升式光生物反应器用于转基因海带配子体的高效增殖。研究发现，气升式光生物反应器较鼓泡式光生物反应器能明显地改善反应器内的传质状态，实现转基因海带配子体更高密度的培养（生物量可达到1,990 mg L-1），是一套高密度悬浮培养转基因海带配子体的有效装置和设备。

利用海带配子体营养增殖体系实现外源基因表达

1993年以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，利用海带世代交替生活史，建立了海带遗传转化体系,并申请国家专利PCT/CN03/00534（Trends in Biotechnology, 2005, 23,264-268.）。海带遗传转化体系是建立在基因枪转化海带配子体，经孤雌或受精途径再生幼孢子体后用氯霉素筛选以及海上安全栽培等一系列技术平台上的新型体系。目前该体系已经成功实现报告基因（β-半乳糖甘酶基因，LacZ）和目的基因（乙肝表面抗原基因，HBsAg）在孢子体阶段的稳定表达。由于应用该体系表达目的基因须经过孢子体再生以及海上栽培等阶段，周期较长，因此本论文拟通过以下5方面工作初步建立以配子体营养增殖为基础的新型海带表达体系，并尝试在配子体阶段实现转化、筛选、增殖及产物检测等操作。1)确立针对配子体的选择标记。2)利用光生物反应器增殖配子体。3)拓展新的报告基因种类。4)克隆内源高效启动子。5)利用该体系表达rt-PA基因（编码价格昂贵的特效溶栓药，瑞替普酶）。本论文研究结果为1)升高温度能够显著增强草丁膦的筛选效果，根据敏感性实验统计结果确定了草丁膦作为海带配子体遗传转化选择压力的筛选方案为40mg/l连续处理7d×3次。2)利用室内光生物反应器营养增殖海带配子体技术，经草丁膦筛选，实现了SV40启动子驱动草丁膦抗性基因bar在配子体细胞中的稳定表达。3)采用调整配子体营养生长状态的方法减低背景荧光，从而首次观察到增强型绿色荧光蛋白基因（egfp）在海带配子体细胞中的瞬间表达。4)克隆了海带肌动蛋白基因（actin）的基因组序列，这是褐藻肌动蛋白基因组序列克隆的首次报道，通过染色体步移获得海带actin基因上游约450bp的片段。5)在上述工作基础上，与中国药科大学合作构建了瑞替普酶基因（rt-PA）的海带表达载体，并实现该基因在海带配子体细胞中的稳定表达，产物具有正确的生物学活性，平均表达量达到0.159μg/mg可溶蛋白。这是该基因在植物中表达的首次报道。研究结果提示在光生物反应器营养增殖体系基础上构建海带配子体表达系统的方案是可行的。

重组竹叶青蛇毒纤溶酶原激活剂TSV-PA效应观察体内溶栓

】目的观察重组竹叶青蛇毒纤溶酶原激活剂一体内溶栓效应。方法利用家兔制备的脑血检 模型, 以生理盐水和尿激酶为对照, 用数字减影血管造影技术观察重组竹叶青蛇毒纤溶酶原激活剂飞体内溶 检效果。结果昭高剂量实验组喇再通时间为以内月州低剂量实验组止以内再 通。尿激酶组以刃再通时间为一以内生理盐水对照组没有观察到再通现象。实验动物后处 死解剖, 未观察到出血现象和其它异常现象。结论重组巧勺一在体内具有良好的溶检活性。

B itte r an d Sw e e t /Um am i Ta s te Re c ep to rs w ith D iffe ren tly Evo lu tio n a ry Pa thw ays

通过生物信息学和系统发育学分析,研究了苦味受体和甜味/鲜味受体的进化途径。结果显示,苦味受体 和甜味/鲜味受体在进化上具有远相关,并且具有不同的进化途径,提示这可能是导致这些受体具有不同功能,传 导不同味觉的原因。

Synthesis and biological evaluation of N-acetyl-beta-aryl-1,2-didehydroethylamines as new HIV-1 RT inhibitors in vitro

A variety of N-acetyl-o-aryl-1,2-didehydroethylamines were synthesized by direct reduction-acetylation of beta-aryl-nitroolefins and assayed as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) for the first time. Compound 7a exhibited a TI v

RT-PCR法检测污染物甲基汞对调钙质基因表达的抑制

应用RT -PCR方法 ,通过用不同浓度的甲基汞对Hep2B细胞株进行不同时间长短的处理 ,检测其调钙质的mRNA表达水平 ,发现微量的甲基汞处理就可以引起细胞株细胞损伤 ,同时抑制调钙质mRNA的表达 ,进一步证实了在肝脏损伤的病理状态下调钙质基因的表达减少 ,从而为检测环境中甲基汞的含量提供了一个可能的分子生物学手段 .

微生物降解邻苯二甲酸(O-PA)的动力学

<正> 苯二甲酸及其脂类在石油化工,特别是作为合成工业的增塑剂和韧化剂和在染料制造业中的重要性以及他们对江河湖海的广泛污染而越来越引起人们的关注。Kluwe研究了邻苯二甲酸脂的致癌影响。Kozumbo发现了这种脂类的致突变作用。Kaneshima发现,苯二甲酸浓度在500—1000ppm时毒性甚大;Wilson指出,苯二甲酸钠在500ppm以上时有很大毒性。所以,关于苯二甲酸在天然环境里归趋的研究便相继进行。Ribbons等用测压技术研究了土壤假单胞菌类对苯二甲酸的氧化代谢,证明4,5-二羟基苯二甲酸为其

Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR

A rapid, sensitive and highly specific detection method for Aquareovirus based on reverse-transcription polymerase chain reaction (RT-PCR) was developed. Based on multiple sequence alignment of the cloned sequences of a local isolates, the Threadfin reovirus (TFV) and Guppy reovirus (GPV) with Grass carp reovirus (GCRV), a pair of degenerate primers was selected carefully and synthesized. Using this primer combination, only one specific product, approximately 450 bp in length was obtained when RT-PCR was carried out using the genomic double-stranded RNA (dsRNA) of TFV, GPV and GCRV. Similar results were also obtained when Chum salmon reovirus (CSRV) and Striped bass reovirus (SBRV) dsRNA were used as templates. No products were observed when nucleic acids other than the dsRNA of the aquareoviruses described above were used as RT-PCR templates. This technique could detect not only TFV but also GPV and GCRV in low titer virus-infected cell cultured cells. Furthermore, this method has also been shown to be able to diagnose GPV-infected guppy (Poecilia reticulata) that exhibit clinical symptoms as well as GPV-carrier guppy. Collectively, these results showed that the RT-PCR amplification method using specific degenerate primers described below is very useful for rapid and accurate detection of a variety of aquareovirus strains isolated from different host species and origin. (C) 2004 Elsevier B.V. All rights reserved.

Radar interferometer measurements of space debris using the Evpatoria RT-70 transmitter

The ability of the Evpatoria RT-70 radar complex to perform research on space debris was investigated in four trial experiments during 2001-2003. The echo-signals of 25 objects at geostationary, highly elliptical and medium-altitude orbits were recorded on magnetic tapes at radio telescopes in Russia, Italy, China and Poland. The multi-antenna system configuration gives potential to supplement the classic radar data with precise angular observations using the technique of Very Long Baseline Interferometry. The first stage of such processing was fulfilled by the correlator in N. Novgorod, Russia. The cross-correlation of transmitted and received signals was obtained for the 11 objects on the Evpatoria-Bear Lakes, Evpatoria-Urumqi and Evpatoria-Noto baselines. This activity also promoted developing the optical observations of geostationary objects, conducted for the improvement of the radar target ephemerides. (C) 2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

The influence of oxygen content on photoluminescence from Er-doped SiOx

SiOx films with oxygen concentrations ranging 13-46 at.% were deposited by plasma enhanced chemical vapor deposition (PECVD) technique using: pure SiH4 and N2O mixture. Erbium was then implanted at an energy of 500 KeV with dose of 2x10(15) ions/cm(2). The samples were subsequently annealed in N-2 for 20 sec at temperatures of (300-950 degrees C). Room temperature (RT) photo-luminescence (PL) data were collected by Fourier Transform Infrared Spectroscopy (FTIS) with an argon laser at a wavelength of 514.5 nm and an output power from 5 to 2500 mw. The intense room-temperature luminescence was observed around 1.54 mu m. The luminescence intensity increases by 2 orders of magnitude as compared with that of Er-doped Czochralski (CZ) Si. We found that the Er3+ luminescence depends strongly on the SiOx microstructure. Our experiment also showed that the silicon grain radius decreased with increasing oxygen content and finally formed micro-crystalline silicon or nano-crystalline silicon. As a result, these silicon small particles could facilitate the energy transfer to Er3+ and thus enhanced the photoluminescence intensity.