人类一条新的类蛋白质二硫键异构酶cDNA的克隆和表达

利用SMART技术构建了人胎脑cDNA文库，通过大规模测序筛选出一条1645bp的人类cDNA。此cDNA含有一个888bp的开放阅读框（ORF），编码一个296个氨基酸的蛋白质，预测分子量34．0KD。与目前数据库中序列比较，该cDNA编码的蛋白质与蛋白质二硫键异构酶的同源性达36％，命名为类蛋白质二硫键异构酶（PDI－L）基因，多组织Northern blot分析显示PDI－L cDNA在心、脑、肝肾等组织中均有表达，该cDNA的读框片段正确插入到改造后的pBV220表达载体中，获得了预期的表达蛋白。     

应用长链RT-PCR法扩增我国登革2、4型病毒株全长cDNA

目的：采用长链RT-PCR技术扩增登革2型及4型病毒基因组全长cDNA，为构建登革病毒全长cDNA克隆、表达，深入阐明致病机理及探索新型疫苗奠定基础。方法：根据已测定的登革2、4型病毒全基因组序列，设计上下游引物。从感染登革病毒的乳鼠脑中提取病毒基因组RNA，采用长链RT-PCR技术进行扩增。为检验扩增产物的特异性，以PCR产物为模板扩增覆盖基因组的10个片段。将含有复杂二级结构的5′非编码区扩增片段，在377A型自动测序仪进行序列分析。结果：扩增出登革2、4型病毒基因组全长近11kb cDNA分子，非编码区测序结果表明扩增产物为登革2、4型病毒所特有。结论：利用长链RT-PCR首次成功扩增出登革病毒全长cDNA分子。     

白纹伊蚊和埃及伊蚊defensin A基因克隆及序列分析

应用PCR技术从白纹伊蚊和埃及伊蚊基因组中扩增出defensinA基因 ,并与文献报道的defensinA的5个型的cDNA序列进行同源性比较 ,发现此两序列中存在内元 ;从埃及伊蚊体内扩增的片段为蚊虫defen sinAl的前体AaDefAl;从白纹伊蚊体内扩增的片段为defensinA的 1个新型 ,命名为DefA6。     

CLONING AND EXPRESSION OF cDNA FOR HUMAN LYMPHO-TOXIN

人淋巴毒素（ｈＬＴ）系由淋巴细胞经抗原或有丝分裂原活化后产生的一类细胞因子，它具有抗瘤、抗病毒活性和许多重要的免疫调节作用，是一种非常有前途的生物制剂。近年来发现的膜相关型淋巴毒素更提示ｈＬＴ可能具有尚未被揭示的免疫调节活性。因此，克隆人ＬＴｃＤＮＡ并在大肠杆菌表达重组ｈＬＴ，对于ｈＬＴ的开发利用和研究其功能都具有重要意义。本实验按照公布的ｈＬＴｃＤＮＡ序列，经计算机分析并结合实验要求设计并合成一对ＰＣＲ引物，采用ＲＴ－ＰＣＲ技术从ＰＨＡ／ＰＭＡ活化２４ｈ的人Ｔ细胞系Ｊｕｒｋａｔ细胞总ＲＮＡ扩增出一５４１ｂｐＤＮＡ片段；经α－互补筛选，质粒小量快速抽提，限制性内切酶酶切鉴定，将该片段定向克隆于ｐＵＣ１８、ｐＵＣ１９质粒载体。限制性内切酶图谱分析和Ｓａｎｇｅｒ双脱氧链终止法序列测定表明：该ＤＮＡ片段与公布的人淋巴毒素ｃＤＮＡ序列完全一致。它包括编码人淋巴毒素成熟肽的全部ｃＤＮＡ序列。再进一步将该ｃＤＮＡ片段克隆于原核表达载体ｐＢＶ２２０，经地高辛标记探针菌落原位杂交筛选，限制性内切酶酶切鉴定方向，筛选出一阳性重组子ｐＢＶ－ｈＬＴ。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析表明：经温控诱导，该重组菌成功     

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

人硫氧还蛋白cDNA的克隆、表达及其多克隆抗体制备

目的制备硫氧还蛋白1(thioredoxin-1,Trx-1)多克隆抗体。方法从乳腺癌细胞系MCF-7中用RT-PCR的方法得到了Trx-1全长基因,将它克隆到原核表达载体上进行大量的表达和纯化,纯化的蛋白对新西兰大白兔进行背部多点注射,40d后取其血清用梯度饱和硫酸铵沉淀的方法进行多克隆抗体的纯化。用ELISA和Western印迹实验测定抗体效果。结果成功获得了Trx-1全长cDNA,通过原核表达得到了大量Trx-1蛋白,并制备了高效价的多克隆抗体。结论此多克隆抗体对Trx-1蛋白具有良好的识别能力,可以应用于Trx-1的功能研究。     

Onset of promiscuous gene expression in murine fetal thymus organ culture

T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15-17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance.     

体外铅染毒对人膀胱细胞基因表达谱的影响

目的研究体外培养的人胚胎膀胱组织来源细胞(humanbladder,HB)在铅暴露不同时间和浓度下的基因表达差异,以探讨铅毒性的作用机制。方法分别使用100、200μg/ml乙酸铅溶液暴露体外培养的HB细胞(浓度为2×103个/ml)8、24h,设置空白对照,提取RNA,进行cDNA基因表达谱芯片实验。芯片扫描结果经归一化处理,设定Ratio值0.5或≥2为表达有差异基因。结果铅暴露HB细胞导致差异表达的基因共有2406个,其中,在铅暴露不同时间(8、24h)、不同浓度(100、200μg/ml)皆上调的有45个基因,皆下调的有40个基因。结论基因表达因铅暴露的时间和浓度变化而发生改变;差异表达基因的功能与信号传导、细胞代谢密切相关,也与疾病发生发展相关;可以通过分析基因表达谱改变,探悉铅暴露导致机体损伤的原因和致病机制。