长白蝮蛇类凝血酶基因的克隆及分析(英文)

从长白蝮蛇 (Agkistrodon halys Ussuriensis)毒腺中抽提总 RNA,采用 RT-PCR扩增其类凝血酶基因 ,经全序列测定 ,类凝血酶基因 Ussurin全长为 70 8个核苷酸 ,即编码 2 3 6个氨基酸 ;根据同源性 ,推测它的活性中心为 His43,Asp88和 Ser182 ;二硫键为 Cys7-Cys14 1,Cys2 8-Cys44,Cys76 -Cys2 34 ,Cys12 0 -Cys188,Cys152 -Cys16 7和 Cys178-Cys2 0 3.该蛇毒类凝血酶 c DNA序列及推导的氨基酸序列均为首次报道 .

长白山赤杨丛枝菌根真菌多样性的半巢式LP-PCR-SSCP检测分析

利用半巢式LP PCR SSCP技术 ,对中国吉林长白山不同海拔生境下 3种赤杨共生丛枝菌根真菌的多样性进行检测分析 .结果表明 ,该地区赤杨属东北赤杨、西伯利亚赤杨及色赤杨共生丛枝菌根真菌在科乃至种的水平上并未随宿主的变化表现出丰富的多样性 ;3个树种在自身属的水平上与共生的球囊霉科 (Glomaceae)至少 1种AMF ,即G .intraradix ,在种的水平上表现出不相关于宿主海拔高度的某种相互选择性 .

长白山四种赤杨丛枝菌根真菌侵染多样性的巢式PCR-RFLP分析

采集中国吉林长白山不同海拔的4种赤杨根须样本,利用巢式PCR-RFLP方法检测丛枝菌根真菌(AMF)对样品的侵染情况,PCR结果经限制性内切酶分析.结果表明,赤杨根内AMF存在丰富的基因多样性.AMF的侵染有从宿主混乱性向宿主专一性发展的趋势.东北赤杨AMF的宿主专一性水平最强,球囊霉属已成为东北赤杨的优势侵染类群;其余3种赤杨的AMF则出现宿主混乱现象.宿主因素比海拔因素对AMF侵染有更重要的影响.

镉胁迫对拟南芥幼苗错配修复基因表达的影响

采用半定量反转录聚合酶链式反应(RT-PCR)技术研究了Cd胁迫对拟南芥(Arabidopsis thaliana)幼苗错配修复和增殖细胞核抗原基因表达的影响,并结合幼苗的形态和生理指标,选取Cd胁迫敏感的生物标记物。结果表明:不同浓度(0.25、0.5、1.0 mg.L-1)Cd处理对拟南芥幼苗叶片数、地上部鲜质量影响不大;Cd浓度为0.25 mg.L-1时,地上部分可溶性蛋白质含量明显升高(P<0.05),Cd浓度为0.5和1.0 mg.L-1时,可溶性蛋白质含量明显降低(P<0.05);叶绿素含量随着Cd浓度的增加而微弱增加(P>0.05)。Cd浓度为0.25 mg.L-1时,以18S rRNA为内参照,PCNA1、PCNA2、MSH2、MSH3、MSH6、MSH7 6个基因均出现了诱导表达,当Cd浓度增加到1.0 mg.L-1时,除了MSH6持续表达诱导及MSH3基因与对照相比表达抑制外,其他基因的表达依然出现诱导,但都低于0.5 mg.L-1Cd处理下的基因表达水平。以上结果表明,基因表达的改变可作为检测Cd污染对植物遗传毒性效应潜在有用的生物标记物。

几种PCR方法在克隆鸭肠炎病毒未知基因中的应用

以DEV基因组DNA为模板,用简并PCR、改良Targeted gene walking PCR、改良的热不对称交错PCR和Long-PCR,获得了5350bp、11083bp和2905bp3段DEV未知基因片段,DNA序列分析发现包含9个开放阅读框,将这些序列提交GenBank分别获得的登录号为:EF554396~EF554403。结果表明,多种PCR方法联合使用可以高效的实现对鸭肠炎病毒未知基因的克隆。

利用rep-PCR研究云南尼泊尔桤木根瘤内Frankia基因多样性

利用rep-PCR指纹技术对云南5个地区的尼泊尔桤木根瘤内Frankia菌基因多样性进行研究,实验发现Frankia菌呈现丰富的基因多样性.所有71个样品被分为11种不同的基因类型.除高黎贡山外,不同地域都有某种基因型占优势,显示Frankia菌基因型与地域有紧密关系.所观察的5个地区中,高黎贡山Frankia菌基因型种类最多,多样性指数最高,基因多样性最为丰富.结合高黎贡山地理资料及其它生物多样性相关研究,推测与高黎贡山尼泊尔桤木共生的Frankia可能作为种储备库,为其它地区的寄主植物提供了祖先菌株.图8表1参13

实时荧光定量PCR及其在微生物生态学中的应用

定量描述微生物群落的组成,在微生物生态学的许多研究领域都是非常重要的。然而由于可培养技术的局限性,定量描述微生物群落成为比较困难的事情。最近包括PCR技术在内的分子生物学技术为人们提供了有力的工具,使对微生物群落的分布、丰度等有了进一步的了解。实时荧光定量PCR技术作为核酸定量检测技术,自从发明以来在微生物生态学研究中逐渐得到了广泛的应用。从微生物生态学角度,综述了实时荧光定量PCR技术的原理、发展、优缺点及其在微生物生态学研究中的应用与研究进展,并探讨了实时荧光定量PCR技术的发展和应用前景。

用改进的重叠PCR引入血管内皮生长因子基因突变

血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)的PCR产物克隆于T载体上 ,经转化JM1 0 9感受态菌株后 ,随机挑取 8个白斑菌落 ,混合后制成混合模板 .采用 3条引物 ,做两轮重叠PCR反应 ,获得了VEGF的突变基因 ,经PCR鉴定 ,酶切鉴定和测序分析表明所得基因为目的产物 .实践证明这种突变方法简单快速 ,为下一步实验大量引入突变奠定了实验基础

大鼠脑胶质瘤干细胞分离及作为树突状细胞治疗抗原来源的研究

脑胶质瘤是原发性恶性脑肿瘤中最常见的一类肿瘤，其具有较差的预后性和高致死率。尽管包括手术、化疗及放疗在内的综合治疗水平在不断提高，但由于胶质瘤细胞的高度侵袭性导致治疗结果仍未得到明显改善，病人常常在治疗后很短时间内发生复发。近年来在肿瘤发生的领域提出了一个新的理论，即肿瘤干细胞理论。这个理论认为，肿瘤干细胞是肿瘤中具有自我更新、无限增殖及分化潜能的细胞，这部分细胞虽然只占少部分，但却是肿瘤发生、发展的关键。本研究主要探讨了利用化疗药物秋水仙素分离大鼠胶质瘤细胞中的干细胞样肿瘤细胞（Stem-like Cancer Cell, SLCC）的可行性，负载胶质瘤SLCC的树突状细胞疫苗在体外对肿瘤细胞的杀伤能力，以及对秋水仙素处理胶质瘤细胞的基因组不稳定性的初步探索。 C6大鼠胶质瘤细胞经不同剂量水平（0、0.5、1.0、2.0μg/ml）的秋水仙素处理后，大部分细胞发生凋亡，剩下存活的细胞经过免疫荧光染色发现其表达干细胞标志分子Nestin，提示可能是SLCC。流式和RT-PCR实验表明，经过秋水仙素处理后的存活的C6细胞表达干细胞标志分子nestin，sox2和bmi1，并且其数量随着秋水仙素浓度的增加而增加。 树突状疫苗是一种脑肿瘤的生物治疗方法。本实验通过分离大鼠的树突状细胞和淋巴细胞，体外培养并用经不同剂量组处理分离的SLCC全蛋白刺激一周后，使用MTT法检测激活的细胞毒T淋巴细胞（cytotoxic T lymphocyte，CTL）体外对肿瘤细胞的杀伤效果。结果表明，经药物分离的SLCC组诱导的CTL效果明显大于对照组，提示脑胶质瘤SLCC可以作为树突状疫苗免疫治疗的新靶点。 此外RT-PCR证明经秋水仙素处理后的胶质瘤细胞中mad2水平明显升高，表明化疗药物可能增加了肿瘤细胞中基因组不稳定性，从而导致SLCC的聚集。 综上，本实验证明了利用化疗药物秋水仙素可以分离大鼠胶质瘤细胞C6中的SLCC细胞，且该分离可能与肿瘤细胞基因组不稳定性提高有关。同时，利用负载SLCC全蛋白的树突状细胞激活的T淋巴细胞在体外具备比对照组更强的细胞杀伤能力。这些结果为以脑胶质瘤SLCC最为靶点的免疫治疗方法开发提供了基础依据。

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

虎眼万年青多糖对小鼠免疫功能的调节作用

依据虎眼万年青组分中多糖对小鼠巨噬细胞吞噬功能的影响,筛选并优化出60%醇浓度中性多糖活性组分S3,进而研究其对体液免疫、细胞免疫及细胞因子变化的影响,并从细胞和分子水平探讨其作用的机理.方法:采用溶血素测定方法,测定S3对小鼠脾细胞溶血素抗体的诱导作用;采用3H-TdR渗入法,测定S3对ConA诱导小鼠脾淋巴细胞的增殖作用;采用3H-TdR后标记法,测定S3对NK细胞细胞毒活性的影响;以ELISA法测定S3对小鼠脾细胞IL-2产生的影响;利用流式细胞术,检测S3对T淋巴细胞亚群CD3、CD4、CD8、CD4/CD8阳性细胞百分率的影响;采用RT-PCR方法检测IL-2 mBNA的表达水平.结果:S3高、中剂量组能明显增强小鼠脾细胞溶血素抗体的形成(P<0.05);各剂量组均能明显增强ConA诱导的淋巴细胞增殖能力(P<0.001);各剂量组均能增强NK细胞的细胞毒活性并促进IL-2的产生(P<0.001,P<0.01);高、中剂量组CD8阳性细胞百分率明显降低(P<0.001),各剂量组均能显著地提高CD4/CD8阳性细胞百分率(P<0.001);高剂量组可明显促进脾细胞中细胞因子IL-2的mRNA表达,使表达量增加(P<0.05).结论:S3具有较强的增强机体多种免疫功能的作用,可利用开发为一种免疫增强药物.

Progress in electrochemiluminescent analysis

The recent progress in electrochemiluminescent (ECL) assay is reviewed. This review begins with the fundamental researches in ECL, including the discovery of new ECL-active species, such as biochemical, organic and metallorganic materials, digital modeling of ECL process, the flow cells used in ECL assay, and electrochemiluminescent sensor. The application of ECL in environmental analysis, immunoassay, nucleotide acid hybridization sensor. The applications of ECL in environmental analysis, immunooassay, nucleic acid hybridization assay, and other aspects are reviewed with the latest references in detail. Finally, the main problems in the further investigation are outlooked, so are its prospects.

The involvement of HSP22 from bay scallop Argopecten irradians in response to heavy metal stress

Heat shock protein 22 (HSP22) is an important member of small heat shock protein (sHSP) subfamily which plays a key role in the process of protecting cells, facilitating the folding of nascent peptides, and responding to stress. In the present study, the cDNA of HSP22 was cloned from Argopecten irradians (designated as AiHSP22) by rapid amplification cDNA end (RACE) based on the expressed sequence tags (ESTs). The full-length cDNA of AiHSP22 was of 1,112 bp, with an open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd2+, Pb2+ or Cu2+ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a 10 days exposure of scallops to Cu2+, Pb2+ and Cd2+. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. Different concentrations of the same metal resulted in different effects on AiHSP22 expression. The sensitive response of AiHSP22 to Cu2+, Pb2+ and Cd2+ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

P>NF-kappa B is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-kappa B-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor kappa B (I kappa B)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-kappa Bs and phylogenetic analysis suggested that EsRelish was a member of the NF-kappa B family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.