A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression.     

Kinetics of lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reversed micelles

The kinetics of lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reversed micellar media was studied. It was shown that the deactivation of lipase had a great influence on the reaction kinetics. Based on whether the enzyme deactivation and influences of both product and substrate on enzyme stability were included or not, four different kinetic models were established. The simulating results demonstrated that the kinetic model, which including product inhibition, enzyme deactivation and the improvements of lipase stability by both product and substrate, fit the experimental data best with an overall relative error of 4.68%.     

LGT蛋白质组阳性肿瘤患者免疫功能的观察报告——LGT系列论文之八

目的：了解LGT（lost goodwill target）蛋白质组阳性表达患者CD3^＋、CD4^＋、CD8^＋、CD4^＋/CD8^＋、T细胞和NK细胞的变化规律.方法：对30例LGT蛋白质组阳性表达的肿瘤患者分别采用美国BD公司生产的流式细胞检测仪及提供的相应单克隆抗体检测患者空腹血清CD3^＋、CD4^＋、CD8^＋、CD4^＋/CD8^＋、T细胞和NK细胞并用美国赛费吉（Ciphergen）公司制造的蛋白质指纹仪及该公司提供的弱阳离子交换芯片（WCX2）按其操作方法（SELDI检测技术）配对检测肿瘤患者空腹血清中的蛋白质指纹,对有病情加重的患者均检查两次以上.以指纹图上质荷比（M/Z）为11100＋H～11900＋H之间出现一峰簇样（cluster）的指纹标志为LGT阳性诊断标准,并按蛋白质指纹LGT检测阳性次数分成二组.对二组内CD3^＋、CD4^＋、CD8^＋、CD4^＋/CD8^＋、T细胞和NK细胞与正常参考值之间进行统计学显著性检验.结果：CD3^＋T细胞值在LGT蛋白质组持续阳性表达组是增高的,在另一组是无变化,二者之间有显著性差异,而CD8^＋T细胞在二组内同时增高,CD4^＋T细胞和NK细胞二组同时低下,无显著性差别.结论：本研究提示肿瘤晚期可能存在有酪氨酸蛋白激酶修饰的细胞内信号传导,使之病情加重,而肿瘤早期则不明显.这是一种新的看法,应加强这个方面的研究.     

Orchard floor management practices that maintain vegetative or biomass groundcover stimulate soil microbial activity and alter soil microbial community composition

Groundcover management systems (GMS) are important in managing fruit-tree orchards because of their effects on soil conditions, nutrient availability, tree growth and yields. We employed a polyphasic approach, incorporating measures of soil microbial abundance, activity and community composition, to study the long-term effects of different GMS on biotic and abiotic factors in an orchard soil. Four GMS treatments – Pre-emergence residual herbicides (Pre-H), post-emergence herbicide (Post-H), mowed-sod (Grass), and hardwood bark mulch (Mulch) – were established in 2-m-wide strips within tree rows in an apple orchard in 1992, and have been maintained and monitored annually until the present. We have measured soil water and nutrient availability, tree growth, and yields annually from 1993 to 2003. Soil nematode numbers and trophic groups were evaluated in July and Oct. 2001, and Sept. 2003. Numbers of culturable bacteria and fungi, soil respiratory activity, eubacterial and fungal community composition were determined in May and Sept. 2003. The Pre-H treatment soil had the fewest culturable bacteria, while the Grass treatment had the largest population of culturable fungi. Soil nematode population size and diversity were also affected by GMS treatments; the Pre-H treatment had the lowest ratio of (bacteriovores + fungivores) to plant parasitic nematodes. Soil respiration rates were higher in the Mulch than in other treatments during a 40-day incubation period. Hierarchical cluster dendrograms of denaturing gradient gel electrophoresis (DGGE) fingerprints for eubacterial community 16S rRNA genes indicated that Post-H and Grass treatments clustered together and separately from the Pre-H and Mulch treatments, which were also grouped together. The influence of GMSs on the fungal community, as assessed by PCR-DGGE of the internal transcribed spacer (ITS) region, was not as pronounced as that observed for bacteria. Soil fungal community composition under the Mulch differed from that under other treatments. The effects of GMS on soil microbial community abundance, activity, and composition were associated with observed differences in soil organic matter inputs and turnover, nutrient availability, and apple tree growth and yields under the different GMS treatments.     

RUS家族对植物生长发育的调控作用

叶绿体基因组编码许多参与光合作用和其他代谢过程的关键蛋白质,在叶绿体中合成的代谢物对于植物正常的生长发育至关重要。根对紫外线-B辐射敏感[Root-UVB (ultraviolet radiation B)-sensitive, RUS]蛋白属于叶绿体蛋白,由高度保守的DUF647结构域组成,在参与植物形态发生、物质运输和能量代谢等多种生命活动的调控中发挥作用。本文就近年来关于RUS家族在植物的胚胎发育、光形态建成、维生素B6稳态、生长素转运和花药发育等生长发育过程中的相关研究进行回顾和总结,为深入研究其在植物生长发育中的分子调控机制提供了参考。     

低能离子束介导转基因技术

通过与常规的植物转基因方法进行比较,阐述了低能离子束介导转基因的原理和优点,以及该技术自开创以来在应用领域中取得的成果。     

对虾白斑综合症病毒囊膜蛋白VP28的表达及其抗病毒感染作用

根据GenBank上WSSV囊膜蛋白基因vp28的序列,设计并合成引物,PCR扩增得到vp28基因,成功构建重组表达载体pET22b-vp28并转化大肠杆菌BL21(DE3)。基因工程菌株37℃IPTG诱导,表达产物经Western-blot和SDS-PAGE检测显示有与预期大小32kDa相符合的目的蛋白。用Ni2 -柱纯化的目的蛋白分别直接注射螯虾和包被饲料投喂螯虾,实验结果表明vp28在大肠杆菌中的表达产物有显著提高虾体抗WSSV感染力的作用,而且注射效果更好。     

Tropic 1808基因重组蛋白对大鼠坐骨神经再生的影响

目的观察Tropic1808基因重组蛋白对坐骨神经损伤后再生的影响。方法SD大鼠16只,分为Tropic1808基因重组蛋白组和生理盐水组,切断左侧坐骨神经,硅胶管套接后两神经断端间距10mm,再生室内注入Tropic 1808基因重组蛋白液(500μg/ml)或生理盐水13μl。术后每4周做一次足迹试验,16周时作神经干动作电位、脊髓前角运动神经元数、腓肠肌纤维截面积、硅胶管中段再生神经等检测。结果Tropic1808基因重组蛋白组SFI的恢复、神经干动作电位、脊髓前角运动神经元数、腓肠肌纤维截面积、硅胶管中段再生神经有髓神经纤维数等结果均明显优于生理盐水组,有统计学意义。电镜观察表明Tropic1808组再生轴突较NS组粗,髓鞘较生理盐水组厚。结论Tropic1808基因重组蛋白有促进大鼠坐骨神经再生的作用。     

未绝经宫颈癌患者肿瘤组织中环氧合酶2的表达及其临床意义

目的探讨环氧合酶2(COX-2)在宫颈癌组织中的表达与月经周期的关系及其临床意义。方法选取47例未绝经宫颈癌手术病人,病史结合子宫内膜HE染色观察判断患者所处月经周期时段,采用免疫组织化学SP法检测宫颈癌组织中COX-2及增殖细胞核抗原(PCNA)的表达,计算机图像分析COX-2免疫染色密度值及PCNA标记指数(LI)。结果宫颈癌组织中COX-2表达阳性者增生期表达强于分泌期(P<0.05),但阳性率无显著性差异(P>0.05),COX-2阳性癌组织中PCNALI高于阴性者,差异有显著性(P<0.01)。有淋巴结转移者COX-2的表达高于无转移者(P<0.05),宫颈癌组织中COX-2的表达与FIGO临床分期、组织分化程度无显著相关性(P>0.05)。结论未绝经宫颈癌患者肿瘤组织中COX-2的表达在增生期较分泌期明显增加,并参与调节肿瘤的生长、转移。