丝状真菌瑞氏木霉外源基因表达系统的构建

采用PCR技术体外扩增获得了瑞氏木霉外切葡聚糖纤维二糖水解酶Ⅰ (CBHⅠ )启动子和终止子序列 .并以大肠杆菌质粒pUC1 9为骨架 ,在该启动子和终止子序列间加入多克隆位点 ,构建了瑞氏木霉强表达整合型载体pTRIL .以质粒pAN7 1为模板 ,体外扩增了带有潮霉素磷酸转移酶(hph)基因的DNA片段 ,将hph插入pTRIL的cbh1启动子和终止子序列之间 ,构建了Pcbh1 hph Tcbh1表达盒 .用此表达盒转化瑞氏木霉C30原生质体 ,在潮霉素平板上得到 1 5株抗性转化子 .对其中的H1转化子进行了PCR和Southern印迹分析 ,证实hph基因确实整合到转化子染色体DNA上 ,并在Pcbh1 启动子控制下进行高效表达 .转化子H1对潮霉素抗性达 1 5 0mg L ,比出发菌株提高 2倍 .瑞氏木霉强表达整合型载体pTRIL的构建成功为开展瑞氏木霉分子生物学研究以及进一步的工程菌株构建工作奠定了基础     

猪细小病毒单克隆抗体的制备和鉴定

目的制备猪细小病毒（PPV）杂交瘤细胞株,并对其分泌的PPV单克隆抗体进行鉴定。方法按常规方法制备并获得2株杂交瘤细胞。用染色体分析对杂交瘤细胞进行鉴定,用间接ELISA、免疫过氧化物酶单层试验（IPMA）和间接免疫荧光试验（IFA）对其分泌的单克隆抗体进行效价测定、亚型鉴定和特异性鉴定。结果得到2株分泌单克隆抗体的杂交瘤细胞株2H9、1F9,染色体数目介于90～110之间。细胞上清效价均达1∶1×104,腹水效价均达1∶1×107,其亚型分别为IgG1、IgM,均为kappa链。2H9、1F9单抗与猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）、猪圆环病毒I型（PCV-1）、猪圆环病毒Ⅱ型（PCV-2）、乙脑病毒（JEV）等均无交叉反应。IPMA和IFA检测结果显示2H9、1F9单抗均能与接种于PK-15细胞的PPV发生特异性反应。结论成功制备了2株抗PPV杂交瘤细胞株,证实其产生的单克隆抗体具有良好的特异性和敏感性。     

Conditioned medium from hypoxia-treated adipocytes renders muscle cells insulin resistant

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and local inflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environment with the release of pro-inflammatory factors that stimulate macrophage migration and cause muscle insulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxia response by elevating protein expression of HIF-1α. This was associated with elevated mRNA expression and peptide release of cytokines TNFα, IL-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced. Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raised basal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Akt and AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H also caused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes. These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heat inactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulated cell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc traffic through its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversed the inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwell co-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H prevented macrophage migration towards it and partly reversed the effect of CM-H on insulin response in muscle cells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory response observed in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migration towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.     

高等植物开花时程的基因调控(Ⅱ)

主要探讨如下几个问题:转基因植物中的拟南芥开花时程基因;拟南芥中的甲基化与实验胚胎学研究;高等植物开花时程控制的可能机制.目前本领域已成为植物发育分子生物学的前沿热点研究领域之一.结合有关工作,对这一世界性热门领域进行了系统的评述,希望能为国内同行提供有关参考,赶超世界的植物分子生物学先进水平,对分子水平与生物技术角度改良黄土高原生态环境有指导意义.     

噬菌体抗体文库的构建及人源抗HIV-1 gp 160抗体的筛选

构建人源噬菌体抗体文库如下：ＨＩＶ感染者脾细胞中提取ｍＲＮＡ,经反转录再以人ＩｇＧ重链Ｆｄ两端及轻链“通用”引物分别扩增Ｆａｂ基因片段,依次插入到噬菌粒载体ｐＣｏｍｂ３中,电转化大肠杆菌ＸＬ１－Ｂｌｕｅ,经辅助噬菌体救助,重组噬菌体得以溶源裂解,Ｆａｂ表达于噬菌体包膜蛋白Ⅲ的Ｎ端．此噬菌体抗体库的容量为５×１０５．筛选抗ＨＩＶ－１同时又具有能被抗独特型抗体“ＩＦ７”所识别的独特型的阳性抗体：以重组包膜糖蛋白ｇｐ１６０及ｇｐ４１多肽对噬菌体抗体文库进行三次淘选,使抗ｇｐ１６０的特异性抗体得到１００倍的富集．然后通过直接ＥＬＩＳＡ和竞争性ＥＬＩＳＡ实验筛选出两株结合性较好的特异性抗ｇｐ１６０抗体－３Ｂ株与１Ｄ株．直接ＥＬＩＳＡ实验表明这两株克隆均能被“１Ｆ７”所识别,为抗独特型多肽的筛选奠定了基础．     

全反式维甲酸联合替莫唑胺对胶质瘤U251细胞增殖与凋亡的影响

目的：研究全反式维甲酸（ATRA）联合替莫唑胺（TMZ）对胶质瘤细胞株U251增殖及凋亡的影响。方法：体外培养胶质瘤细胞株U251,分为ATRA组、TMZ组、ATRA＋TMZ组和空白对照4组,应用MTT法测定U251细胞生长抑制率,流式细胞仪检测细胞周期分布和细胞凋亡率,westernblot法检测细胞维甲酸受体β（RARβ）蛋白和凋亡相关蛋白Caspase．3蛋白的表达情况。结果：对照组、ATRA组、TMZ组及ATRA＋TMZ组的细胞生长抑制率分别为（1．72±0．12）％、（9．87±0．87）％、（23．87＋1．32）％及（35．74±1．44）％,ATRA＋TMZ组的细胞生长抑制率显著高于单独应用TMZ（P〈0．01）。对照组、ATRA组、TMZ组及ATRA＋TMZ组的细胞凋亡率分别为（1．32±0．11）％、（4．16±0．35）％、（8．44±0149）％、（15．27±1．03）％,ATRA＋TMZ组的凋亡率显著高于单独应用TMZ有更高的凋亡率（P〈0．01）。对照组、ATRA组、TMZ组及ATRA＋TMZ组RARp蛋白相对表达量分别0．452±0．054、0．837±0．068、0．195±0．021、0．376±0．039,ATRA＋TMZ组RARβ蛋白相对表达量显著高于TMZ组（P〈0．01）。ATRA＋TMZ组Caspase．的3蛋白表达相对水平为（0．832±0．059）,明显高于ATRA组（0．334±0．041）及TMZ组（0．521＋0．032）,差异具有统计学意义（P〈0．01）。结论：全反式维甲酸联合替莫唑胺能更有效抑制U251细胞的增殖,增加其凋亡率,这可能与其增加RARβ和Caspase-3蛋白的表达抑制U251细胞增殖、诱导细胞凋亡有关。     

体内精原干细胞转染法建立转基因小鼠

将人Bcl-2 cDNA与小鼠乳清酸蛋白(WAP)5’上游调控序列融合后,与脂质体按一定比例混合,再加入适量的台盼蓝制成转染液,注入到小鼠睾丸中的曲细精管中,转染精原干细胞以探讨建立转基因小鼠的可行性。共注射了3只公鼠,4天后将公鼠与发情母鼠合笼交配,共生仔鼠20只。检测结果表明,有3只呈PCR阳性,Southern blot检测,阳性鼠2只,1只公鼠,1只母鼠,其中,公鼠意外死亡;Western blot证实,1只母鼠的乳腺组织表达了Bcl-2蛋白,其F1代的16只小鼠中,有7只呈PCR阳性。证实了体内精原干细胞转染建立转基因动物的可行性。     

蜡梅17个品种过氧化物同工酶的研究

采用聚丙烯酰胺凝胶电泳系统,多次进行了蜡梅17个品种的过氧化物同工酶测定。测定结果发现:蜡梅酶谱具有多态特性;蜡梅品种间酶谱差异显著,每个品种均有其特征酶谱,或以其酶带数目、Ff及活性强弱、酶带宽度与其它品种相区别。同时,采用"排序方法"和聚类分析,对蜡酶17个品种酶谱的相似程度进行了研究。研究结果表明,两种方法研究蜡梅品种的酶谱,均可达到定量比较水平,避免单一酶谱谱带确定蜡梅品种的偏阳性,为其品种鉴定与检验提供了新的依据和手段。同时,还表明,聚类分析进行的蜡梅品种酶谱聚类结果,与其形态分类相一致,但与其品种群划分不相吻合,其原因可能与蜡梅品种起源的复杂性和多源性所引起。     

Maize uroporphyrinogen III methyltransferase: overexpression of the functional gene fragments in Escherichia coli and one-step purification

S-Adenosyl-L-methionine: uroporphyrinogen III methyltransferase (SUMT), a key regulatory enzyme, converts uroporphyrinogen III to precorrin-2 in the porphinoids biosynthesis. In this study, the mature SUMT was signified that the maize SUMT precursor encoded by the open reading frame of maize SUMT cDNA was deleted the first 91 amino acids constituting the postulated signal peptide. Several mature SUMT fusion and deletion mutants were conducted. It actively expressed in Escherichia coli that the mature SUMT, or the truncated one deleting the C-terminal extra 52 amino acids based on SUMT sequence comparisons. On the contrary, it expressed as an inclusion body in E. coli that the mature SUMT fusion mutant, the SUMT precursor, or the mature SUMT deleting the N-terminal 36 amino acids including glycine-rich region involved directly in SAM binding. The purified His6-tagged mature SUMT was homodimer with a molecular weight of 34 kDa, as shown by SDS-PAGE, 52 kDa using gel-filtration chromatography, and 79 kDa by dynamic light scattering assay. Red fluorescent compounds were associated with the recombinant mature SUMT which were identified as sirohydrochlorin and trimethylpyrrocorphin by spectroscopic analysis. This association slightly altered the protein secondary structure confirmed by circular dichroism assay.     

由不同细胞核背景引起的小麦细胞质雄性不育系(CMS)叶绿体蛋白质组的变化(简报)

叶绿体和线粒体是高等植物细胞内2种重要的细胞器。由于细胞质雄性不育(CMS)被认为是一种由线粒体基因编码的性状,因此,近10多年来,国内外研究者对线粒体基因组结构与功能、由线粒体基因编码的与CMS相关蛋白的研究积累了大量的资料。与线粒体相比,叶绿体与CMS关系的研究相对滞后。虽然一些研究者在核不育水稻中,观