H5N1亚型禽流感病毒NS1基因的原核表达及其ELISA检测方法的建立

采用RT-PCR技术扩增了禽流感病毒（AIV）A／Goose／HLJ／p46／2003（H5N1）的NSl基因，将其克隆于融合蛋白表达载体pMAL-c2X上，转化DH5α大肠埃希氏菌感受态细胞，经BamHⅠ和HindⅢ双酶切鉴定及序列分析，表明筛选到了重组质粒pc2X-NS1。SDS-PAGE电泳结果显示，重组质粒转化TB1大肠埃希氏菌后，经0．3mmol／L的IPTG诱导，融合蛋白MBP-NS1得到大量表达，融合蛋白以可溶形式存在，分子质量约为67ku。Western-blotting检测结果表明，融合蛋白MBP-NS1能够与H5N1亚型AIV活病毒感染康复鸭血清发生特异性反应，而不能够与H5N1亚型AIV灭活疫苗免疫鸭血清发生反应。试验初步建立了以纯化的融合蛋白MBP-NS1为包被抗原的间接ELISA检测方法，为AIV灭活疫苗免疫家禽与AIV感染家禽的鉴别诊断奠定了基础。     

鸡传染性腔上囊病病毒VP2基因在昆虫细胞中的表达

为了深入研究鸡传染性腔上囊病病毒（IBDV）VP2基因的结构和功能,利用Bac-to-Bac系统研制出含有VP2基因的重组杆状病毒rBac-VP2,将rBac-VP2感染Sf9细胞,并用抗IBDV VP2特异性单克隆抗体经间接免疫荧光试验检测,证实感染重组病毒Sf9细胞能高效表达IBDV VP2基因产物;Western-blotting分析结果表明,VP2基因表达产物的分子质量约为40 ku.     

H7N2禽流感病毒分离株血凝素蛋白全基因的序列分析

为进一步分析H7N2禽流感病毒（AIV）分离株血凝素（HA）基因的特性,参照已发表序列设计了1对引物,采用RT-PCR获得了1条约1.7 kb的DNA片段,测序后进行了同源性比较、HA基因系统发育进化树分析和氨基酸编码分析.结果表明,所测的2个分离株的HA基因全长1 664 bp,编码除信号肽以外的HA蛋白的全部544个氨基酸,其中包括HA1的323个氨基酸,HA2的221个氨基酸.2个分离株HA基因核苷酸序列的同源性为99.4%;与GenBank中AIV标准株A/Afri.Star./Eng-Q/983/79（H7N1）的同源性最高,分别为99.4%和99.0%;与美国A/Chicken/NewYork/13142-5/94（H7N2）株同源性很低（仅65.0%）,而与以色列、意大利H7N2 AIV的同源性较高（为96%～97%）;2个分离株在HA基因进化树中均处于H7亚型AIV的欧亚群系分支内.推导氨基酸的序列分析表明,其HA蛋白裂解位点的氨基酸序列为-GR-GLF-,仅包含1个碱性氨基酸（R-）残基,符合低致病力AIV的基因特征.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Quick detection of sex determining region protein gene in mouse fetuses

AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue.     

河西走廊典型绿洲景观格局比较研究--以张掖、临泽、高台、酒泉为例

以地处河西走廊的张掖、临泽、高台和酒泉等县 /市级行政区为景观单元 ,利用LandsetTM影像资料的目视解译 ,在ArcView 3.1和景观格局分析软件FRAGSTATS的支持下 ,计算各绿洲景观格局指标。结果表明 :荒漠类型在四个绿洲景观组分中占绝对优势 ,其次为水浇地 ,但不同绿洲水浇地所占比例相差较大 ,酒泉最高 ,张掖其次 ,高台最低 ;斑块数、斑块密度、边缘密度和平均斑块面积的差异表明 ,张掖绿洲景观类型最多且斑块粒径小 ,而高台则完全相反 ,体现出单调而粒径粗大的荒漠绿洲特征。     

景观格局分布特征的区域差异分析--以鄂尔多斯高原伊金霍洛旗为例

以内蒙古鄂尔多斯高原伊金霍洛旗为研究区域 ,以TM遥感影像解译的 1998年土地利用图为基础资料 ,将伊金霍洛旗划分为毛乌素沙地亚区、乌审凹地亚区和黄土丘陵亚区。从区域尺度 ,利用多样性、优势度、均匀度、分维数、平均斑块形状指数、平均斑块大小、斑块密度指数和斑块体平均斑块边缘等指数 ,对不同景观亚区的景观格局进行了数量分析。结果表明 ,在景观亚区这一尺度上 ,毛乌素沙地亚区一方面对该旗景观格局起控制作用 ,另一方面该亚区所承受的人类活动强度最大 ,景观的破碎化程度最深。而乌审凹地亚区的景观破碎化程度最小 ,人类活动强度最弱。从景观要素来看 ,无论哪一亚区均以天然草地为景观基质 ;不同类型斑块总的格局特点是 ,受人类影响程度愈深的斑块 ,其破碎化程度愈深 ,斑块的复杂性降低。总体而言 ,天然类型的斑块体 ,其斑块复杂性以乌审凹地亚区最为复杂 ,斑块的破碎化程度则以毛乌素沙地亚区最深 ;而人工斑块类型的变化规律在不同斑块类型之间稍有不同。     

Gene Flow Analysis of Phytophthora porri Reveals a New Species: Phytophthora brassicae Sp. Nov.

Isozyme analysis and sequence analysis of the internal transcribed spacer regions (ITS-1 and ITS-2) and the 5.8S subunit of the ribosomal DNA gene repeat were used to examine whether isolates of Phytophthora porri from Allium and Brassica represent a single homogeneous species. Twenty-six strains of P. porri, 16 strains isolated from the genus Allium, and 10 strains isolated from the genus Brassica, were analyzed using malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), represented altogether by four putative loci (Mdh-2, Idh-1, Idh-2, and Ldh-2). Isozyme analysis revealed that strains isolated from Allium contained five private alleles at three isozyme loci (Ldh-2 ⁸³, Ldh-2 ¹⁰⁴, Idh-1 ¹⁰⁸, Idh-1 ¹¹², and Idh-2 ⁹⁸), whereas six different alleles were observed at four isozyme loci (Ldh-2 ⁸⁵, Ldh-2 ¹⁰⁰, Ldh-2 ¹¹⁴, Idh-1 ¹⁰⁰, Idh-2 ¹⁰⁰, and Mdh-2 ¹¹¹) in strains obtained from Brassica. The heterozygosity at the Ldh-2 locus, differing in allele composition, however, between strains from Allium and Brassica, was present in all strains, indicating that it is probably fixed. Sequence analysis of the ITS regions and the 5.8S subunit showed consistent differences between isolates from Allium and isolates from Brassica. Based on isozyme data, ITS sequence analysis and formerly published differences in restriction enzyme patterns of mitochondrial DNA, morphology and pathogenicity, it was concluded that the isolates of P. porri Foister did not represent a homogeneous species. Isolates from Brassica constitute a distinct species which is described here as P. brassicae sp. nov. It was inferred from isozyme patterns, which were in no case intermediate between the two species, that P. porri and P. brassicae do not hybridize and are reproductively isolated by barriers to gene flow.     

额尔齐斯河流域生态系统格局及变化

本文首先对额尔齐斯河流域气候，地貌，水文等环境因子特征进行了评述，进而阐述了流域生态系统的特征和格局以及在自然和人为干扰作用下的变化。分析结果表明在自然干扰作用下流域生态系统的格局与流域水文情势变化相适应，处于准稳定变化状态，而在人为对流域水文情势扰动下，流域生态系统的格局间接地受到影响。从而产生新的格局，其作用的强度，持久性和生态系统的格局变化程度过错大于大于自然干扰作用下的变化。引额调水工程对额尔齐斯河流域河谷生态系统内部单元格局变化具有重要影响。