黄芩苷诱导大鼠气管上皮细胞Mx1和PKR表达的研究

为了研究黄芩苷能否诱导大鼠气管上皮细胞表达抗病毒蛋白,以体外培养的大鼠气管上皮细胞(tracheaepithelial cells,TECs)为模型,采用Western Blotting方法检测了不同质量浓度(10、20、30μg/mL)和不同时间段(6、12、24h)黄芩苷诱导TECs表达抗病毒蛋白(AVPs)Mx1和PKR的量。结果表明,黄芩苷在3个浓度梯度和3个时间段均可以诱导TECs分泌抗病毒蛋白Mx1和PKR;并在30μg/mL,24h时使TECs所产生的抗病毒蛋白最高。这一结果为体外筛选诱导TECs表达抗病毒蛋白的中药有效成分提供了参考,为进一步研究中药抗病毒机理提供了实验依据。     

A Comparison Between Northern Blotting and Quantitative Real-Time PCR as a Means of Detecting the Nutritional Regulation of Genes Expressed in Roots of Arabidopsis thaliana

Quantitative real-time PCR (qRT-PCR) has become a routine and robust technique for measuring the expression of genes of interest, validating microarray experiments and monitoring biomarkers. However, concerns have been raised over the accuracy of qRT-PCR in China as well as in the rest of the world. We have previously used qRT-PCR to study the response of ANR1 and other root-expressed MADS-box genes to fluctuations in the supply of nitrate, phosphate and sulphate under hydroponic growth conditions. In this study, we have used both Northern blotting and qRT-PCR analyses to confirm the nutritional regulation of MADS-box genes in Arabidopsis thaliana and test whether both technologies produce the same results. The information obtained indicated that the qRT-PCR results are consistent with those obtained by Northern blotting hybridization for all the tested root-expressed MADS-box genes, in response to different nitrate,phosphate and sulphate growth conditions. Furthermore, our novel results showed that the expressions of AGL12,AGL18, and AGL19 were all down regulated in response to S and P re-supply in both qRT-PCR and Northern blotting analyses.     

绵羊透明质酸酶-2的真核表达及鉴定

为了构建绵羊透明质酸酶-2(hyaluronidase 2,Hyal-2)的真核表达载体并实现其在牛乳腺上皮细胞内瞬时表达,根据GenBank中绵羊Hyal-2基因序列(登录号:NM_001009754)设计2对引物,并分别在前段上游和后段下游引物5'端引入HindⅢ酶切位点和BamHⅠ酶切位点,同时在上游引物酶切位点后加入KOZAK序列,进行分段PCR扩增。以扩增的两段产物为共同模板运用重叠(overlapping)PCR技术扩增Hyal-2基因全长。通过连接反应将双酶切并纯化后的目的片段克隆于真核表达载体pEGFP-C1上,并通过脂质体转染法将构建好的真核表达载体转染牛乳腺上皮细胞,运用RT-PCR及Western blotting方法分别从核酸及蛋白质水平验证其表达。经测序分析,目的片段与模板核苷酸同源性为99%,氨基酸同源性为99%,且片段插入方向正确。RT-PCR及Western blotting方法检测均出现目的条带,证明成功构建了绵羊透明质酸酶-2的真核表达载体,并在牛乳腺上皮细胞中获得表达。     

细菌性条斑病侵染水稻抗性相关蛋白研究

水稻细菌性条斑病由Xanthomonas oryzae pv. oryzicola引起,是目前威胁亚洲稻区水稻生产的重要病害之一。以水稻品种9311为研究对象, 通过差异蛋白质组学方法,研究了病菌侵染48 h后水稻叶片的差异表达蛋白质,通过分析比对、质谱分析以及数据库检索,从中选择了7个上调表达的鉴定蛋白,包括4个水稻LRK类基因,2个NBS-LRR 类基因和1个PR-10基因家族成员基因。并设计相应的PCR引物,从水稻cDNA 中获得相关基因片段做探针进行Northern杂交分析,结果显示,上述基因在接种病原菌12 h或48 h后表达量增加,表明这些蛋白质参与了抗病反应。     

应用实时荧光定量PCR测定牛体细胞端粒长度

本研究为了验证应用实时荧光定量聚合酶链反应（quantitative PCR,Q-PCR）测定牛基因组端粒长度的可行性,选取18个不同来源牛耳成纤维细胞抽提基因组DNA为样本,对Q-PCR和经典的Southern印迹法进行了相关性分析。结果显示,Q-PCR测定端粒长度相对T/S为1.16±0.24,Southern印迹法测量端粒平均TRF值为16.99 kb±0.85 kb,两种方法获得的结果相关性分析R2=0.5612(P＜0.01),因此实时荧光定量PCR是一种测定牛基因组端粒长度的可靠的方法。     

猪磷酸二酯酶4B2的原核表达和活性鉴定

使用RT-PCR方法扩增猪磷酸二酯酶4B2基因,将其克隆到表达载体,经PCR和测序鉴定的阳性重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导重组蛋白表达。结果显示,目的基因片段大小为1718 bp,与GenBank公布的猪PDE4B2基因序列同源性为99.7%。表达的重组蛋白以包涵体和可溶性蛋白两种形式存在。Western blotting检测结果表明,蛋白质大小约为66 ku,通过液相检测重组蛋白的cAMP-PDE活性为51.46%。制备的多克隆抗体有良好的特异性,为进一步检测天然蛋白PDE4B2和筛选PDE4B2的特异性抑制剂奠定了基础。     

猪附红细胞体抗原特性分析

从自然感染的猪血液中纯化猪附红细胞体,应用SDS-PAGE和免疫印迹试验对猪附红细胞体抗原蛋白组分进行了初步分析。通过SDS-PAGE分离得到13种蛋白质,其蛋白分子量范围为24~170 kD,分别是120、112、110、108、68.2、58、49.6、41.2、30、29、26.2、25和24.3 kD。应用免疫印迹试验筛选出了6种特异性蛋白质,分子量分别是108、98.2、68.2、41.2、35和31 kD。     

PRRSV新疆株N基因原核表达条件的优化及蛋白纯化

[目的]获得重组表达质粒pGEX-6P-N在大肠杆菌BL21中高效表达,分析表达蛋白的免疫学活性及纯化后蛋白的浓度.[方法]通过优化诱导剂终浓度和诱导时间来实现融合蛋白的高效表达,使用BCA法测定蛋白浓度,利用Western blotting分析融合蛋白的免疫学活性.[结果]融合蛋白在37℃条件下,经终浓度为1.2 mmol/L的IPPG诱导表达4.5 h后获得了较高的表达水平.纯化后的融合蛋白经BCA法测定蛋白浓度达到0.587 mg/mL.Western blotting试验表明,GST-N蛋白能与抗PRRSV阳性血清发生特异性反应.[结论]为建立N特异性抗体的间接ELISA检测方法及PRRS诊断试剂盒的研制奠定了基础,为新疆PRRS的防控提供了理论支持.     

地高辛标记核酸探针检测重组人白细胞介素1受体颉颃剂中DNA残余量的研究

试验旨在建立重组人白细胞介素1受体颉颃剂(interleukin-1 receptor antagonist,IL-1ra)原液中宿主菌DNA残余量的检测方法。以重组白细胞介素1受体颉颃剂工程菌基因组DNA为模板,优化试验条件,借助超声波手段,制备地高辛标记的核酸探针, 并以此探针进行狭缝杂交及显色反应。结果显示,在24 批次重组人IL-1ra原液中,每人份使用剂量的宿主DNA残余量均小于10 ng。结果表明,该方法灵敏度较高、特异性较强、重复性较好、操作步骤安全且较为方便,可用于重组人IL-1ra原液及半成品的DNA残余检测。     

Salicylic Acid Induced Insensitivity to Culture Filtrate of Fusarium oxysporum f.sp. zingiberi in the Calli of Zingiber officinale Roscoe

Salicylic acid (SA) was used to induce insensitivity in the callus cultures of Zingiber officinale against culture filtrate (CF) of Fusarium oxysporum f.sp. zingiberi. The treatment of callus cultures with SA (10⁴M) prior to selection with CF of the pathogen-increased callus survival. Exogenous application of SA resulted in increased activity of peroxidase and -1,3-glucanase enzymes in the callus cultures. No increase in the activity of phenylalanine ammonia lyase was obtained. Two new protein bands of 97 and 38kDa molecular weights were obtained by SDS-PAGE analysis of soluble proteins extracted from SA-treated calli. The PR-1 monoclonal antibody used for immunodetection of induced proteins cross-reacted with the 38kDa protein band. In vitro antifungal activity of protein extract of calli treated with SA tested against the spores of F. oxysporum f.sp. zingiberi showed significant reduction in spore germination and germ tube elongation. It is concluded that in ginger, SA may result in the induction of resistance to F. oxysporum f.sp. zingiberi by inducing increased activity of peroxidase, -1,3-glucanase and antifungal PR-proteins.