重组人尿激酶原基因的克隆及工程菌的构建与鉴定

利用RT－PCR技术,从人脐带静脉上皮细胞中克隆重组人尿激酶原基因,用表达质粒pET29a构建了重组质粒pET29a/prouk,并转化至大肠杆菌BL21（DE3）中,作为工程菌。经IPTG诱导表达,在46kDa处有一明显表达条带,表达量为约占菌体总蛋白的20％。该菌种在贮存与复苏及传代过程中具有良好的质粒稳定性和表达稳定性。     

基因工程菌的发酵条件研究

为了获得商品化基因工程产品，要求所选用的基因工程菌高效表达重组蛋白．然而，随着发酵规模的扩大，诸多因素影响基因工程菌蛋白的表达．成功的大规模发酵有两个问题是重要的．一是质粒的稳定性，二是高密度发酵．本文从理论上阐明了ｐＨ，温度、溶氧、比生长速率等条件对重组蛋白产生影响的研究进展．     

人PSF真核表达载体构建及其在CHO细胞中的表达

目的是构建人PSF基因真核表达载体pEGFP-N1-PSF,并在检测其在CHO细胞株中的表达情况。应用DNA重组技术和PCR方法从人宫颈癌Hela细胞中扩增PSF基因,插入pEGFP-N1真核表达载体,构建重组质粒pEGFP-N1-PSF并测序鉴定。将pEGFP-N1-PSF瞬时转染CHO细胞,通过Western blot和RT-PCR方法检测PSF的表达,荧光显微镜下观察绿色荧光蛋白表达。结果 CHO细胞转染pEGFP-N1-PSF真核表达载体后,RT-PCR和Western blot实验发现,在RNA和蛋白水平有PSF的表达,在荧光显微镜下可以观察到融合蛋白EGFP/PSF的表达。成功构建pEGFP-N1-PSF真核表达载体,证实其在CHO细胞中可以表达。     

双基因重组质粒及其应用

近年来，随着生物工程技术的发展，人们已成功地将两种不同目的蛋白的基因构建于同一表达载体。这种双基因载体的构建克服了单一目的基因表达载体的不足．已在农业、医药和生物工程基础理论研究中得到广泛应用。     

pEgr1-Trail重组质粒联合电离辐射对MCF-7细胞杀伤效应的实验研究

目的研究p Egr1-Trail重组质粒联合电离辐射对MCF-7细胞的杀伤效应,为增强肿瘤放疗效果提供实验证据.方法应用Annexin V-EGFP/PI双染法检测细胞凋亡;应用酶标仪检测细胞增殖;应用流式细胞仪检测细胞周期.结果不同处理组作用MCF-7细胞后24 h、不同剂量X射线照射后24 h后,各处理组MCF-7细胞的生长抑制率呈上升态势,生长抑制由弱到强的顺序为:2.0 Gy,p Egr1-Trail+0.5 Gy,5.0 Gy,p Egr1-Trail+1.0 Gy,p Egr1-Trail+2.0 Gy,Egr1-Trail+5.0 Gy.各处理组联合2.0 Gy X射线照射后6 h,细胞凋亡率开始上升,48 h达高峰,其中p Egr1-Trail+2.0 Gy组与其他各处理组比较差异具有统计学意义,且细胞凋亡最为显著(P0.05).在细胞周期方面,2.0 Gy X射线照后6 h各处理组S期细胞百分数呈现上升趋势,24 h达高峰;照后24 h,G_2+M期细胞百分数开始上升,且各处理组比较差异均具有统计学意义(P0.05);照后48 h,G_2+M期细胞百分数达到最高,依旧是p Egr1-Trail+2.0 Gy组上升最显著.结论 p Egr1-Trail重组质粒联合电离辐射作用于肿瘤细胞表现出协同杀伤效应,促凋亡作用强于单独X射照射组或p Egr1-Trail基因干预组.     

人细胞周期蛋白cyclin E基因的克隆与真核表达载体的构建

目的:为研究细胞周期蛋白在肿瘤形成过程的分子机制,构建带FLAG标签的细胞周期蛋白E的真核表达载体,并检测其在瞬时转染HeLa细胞株中的蛋白表达。方法:通过RT-PCR扩增cyclinE基因编码cDNA,并将扩增的cDNA片段插入p3XFLAG-CMVTM-14真核表达载体,重组子经酶切分析和测序鉴定后,用脂质体介导的基因瞬时转染法,将重组正确的表达载体转染HeLa细胞,用Western-blot技术检测其在HeLa细胞中融合蛋白的表达。结果:经酶切鉴定和测序分析证实人cyclin E的真核表达载体构建成功,并能在瞬时转染的HeLa细胞中表达。结论:成功构建了人cyclin E的真核表达载体,为进一步研究细胞周期蛋白的功能奠定了基础。     

大熊猫肠道菌抗生素抗性菌转座酶的基因克隆和表达

对大熊猫肠道大肠杆菌抗生素抗性质粒pAm08CD7339全序列的生物信息学分析结果表明,该质粒中存在一个编码转座酶的DNA片段,两条DNA链均可编码转座酶,其编码区长度分别为717 bp和600 bp,编码238个和199个氨基酸残基（分别命名为Transposase238和Transposase199）.以pAm08CD7339质粒DNA为模板,利用PCR方法对2个转座酶基因进行扩增,构建相应表达质粒,转化不同大肠杆菌菌株进行表达,结果表明：Transposase199可以在E.coli BL2     

Fast acceleration of “killer” electrons and energetic ions by interplanetary shock stimulated ULF waves in the inner magnetosphere

Energetic electrons and ions in the Van Allen radiation belt are the number one space weather threat. Understanding how these energetic particles are accelerated within the Van Allen radiation belt is one of the major challenges in space physics. This paper reviews the recent progress on the fast acceleration of "killer" electrons and energetic ions by ultralow frequency (ULF) waves stimulated by the interplanetary shock in the inner magnetosphere. Very low frequency (VLF) wave-particle interaction is considered to be one of the primary electron acceleration mechanisms because electron cyclotron resonances can easily occur in the VLF frequency range. Recently, using four Cluster spacecraft observations, we have found that, after interplanetary shocks impact the Earth’s magnetosphere, energetic electrons in the radiation belt are accelerated almost immediately and continue to accelerate for a few hours. The time scale (a few days) for traditional acceleration mechanisms, based on VLF wave-particle interactions to accelerate electrons to relativistic energies, is too long to explain our observations. Furthermore, we have found that interplanetary shocks or solar wind pressure pulses, with even small dynamic pressure changes, can play a non-negligible role in radiation belt dynamics. Interplanetary shocks interaction with the Earth’s magnetosphere manifests many fundamental space physics phenomena including energetic particle acceleration. The mechanism of fast acceleration of energetic electrons in the radiation belt responding to interplanetary shock impacts consists of three contributing parts: (1) the initial adiabatic acceleration due to strong shock-related magnetic field compression; (2) followed by the drift-resonant acceleration with poloidal ULF waves excited at different L-shells; and (3) particle acceleration due to the quickly damping electric fields associated with ULF waves. Particles end up with a net acceleration because they gain more energy in the first half of this cycle than they lose in the second. The results reported in this paper cast a new light on understanding the acceleration of energetic particles in the Earth’s Van Allen radiation belt. The results of this study can likewise be applied to interplanetary shock interaction with other planets such as Mercury, Jupiter, Saturn, Uranus and Neptune, and other astrophysical objects with magnetic fields.     

Oxidative damages to DNA by indoor PM10s: Their relationships with trace element compositions

Plasmid DNA assay and ICP-MS analysis were conducted in order to investigate the bioreactivity of inhalable particles (PM10) and the relationship between bioreactivity and trace element compositions of PM10 in Beijing air. A total of four PM10 samples were carefully selected to represent the indoor and corresponding outdoor environments: one from urban smoker's home, two from non-smoker's homes, and the other from the outdoor. In general, the oxidative damage by indoor PM10 was slightly higher than that of outdoor. Among the four sets of samples, the PM10 from the smoker's home had a lowest TD50 (toxic dose of PM10 causing 50% DNA to be damaged), being 100 μg·mL-1, suggesting the highest bioreactivity. The heavy metals are believed to be the main reason for oxidative damage to plasmid DNA. The ICP-MS analysis combined with the DNA assay showed that the water-soluble zinc levels had better relationship with TD50 values than other elements, implying that water-soluble zinc might play an important role in the damage of DNA. It is concluded that the PM10 in smoker's home had the highest level of water-soluble zinc as well as the lowest TD50 (highest bioreactivity). Iron is considered to be one of the most bioreactive elements, but it will cause little damage to plasmid DNA, probably because iron is mainly in water-insoluble state in Beijing PM10.