豚鼠胆汁酸盐输出泵（BSEP）基因克隆及其在胆结石豚鼠肝组织中的表达分析

 目的：克隆并分析豚鼠BSEP基因全长cDNA序列,检测BSEP基因在胆结石豚鼠肝组织中的表达。方法：采用3'、5'RACE方法扩增获得豚鼠肝组织BSEP基因全长cDNA,用生物信息学方法对其进行分析鉴定。实时荧光定量PCR检测BSEP基因在胆结石豚鼠肝组织的表达。结果：豚鼠BSEP基因全长cDNA5579bp,共包含28个外显子,开放性阅读框（ORF）长为3963bp,编码蛋白长1320aa。该基因在胆结石豚鼠肝组织的表达较正常豚鼠显著下调（P<0.01）。结论：豚鼠BSEP基因在胆结石豚鼠肝组织中表达下调可能与结石形成有关。     

小鼠增龄相关性胸腺基因的克隆

目的：用基因差异显示技术研究小鼠胸腺增龄相关基因。方法：利用差异显示反转录PCR（DDRT－PCR）技术对1月龄和10月龄小鼠胸腺mRNA差异表达进行分析,获得差异显示的表达序列标签（ESTs)。选其1个1月龄高表达的EST为探针,从小鼠胸腺cDNA文库中筛选出一个827bp的阳性克隆,并用PCR扩增延长为1406bp的cDNA片段（mt22-1406)。结果：同源性分析结果表明,mt22-1406含一编码438A的开放阅读框,与人延伸因子1γ（EF1γ）高度同源,其Genbank接收号为BE241062。结论：获得一个含完整开放阅读框与小鼠胸腺增龄相关的基因。     

大鼠睾丸精子发生中差异表达基因RSD5的克隆与表达分析

为了分离、克隆精子发生相关基因,深入了解精子发生的分子机理,本文以大鼠睾丸曲线精管显微分离结合DDRT－PCR的方法,所得的不同区段中差异表达的EST（expressed sequence tag)筛选大鼠睾丸cDNA文库,获得一个新的cDNA序列－RSD5。RSD5 cDNA全长1556bp,编码176个氨基酸,GenBank接收号为ASF146738。RSDS基因编码蛋白序列含有一个PEST基序,这是蛋白质快速降解的标志。Northern blot分析结果显示,RSD5在睾丸组织和脑组织表达量较高,且在不同发育天龄的睾丸组织中具有显著的表达差异性。RSD5基因的表达特征及其编码蛋白的PEST基序提示RSD5蛋白可能在精子发生中具有重要作用。     

蛋白质二硫键异构酶A3——疾病治疗的新靶点

<正>蛋白质二硫键异构酶A3(protein disulfide isomerase A3,PDIA3),又称ERp57或ERp57/GRP58,是蛋白质二硫键异构酶(protein disulfide isomerase,PDI)家族的一员,具有催化内质网中蛋白质二硫键形成、氧化还原及异构化的作用。Bennett等[1]于1988年首次从不同物种中分离出PDIA3基因的互补DNA编码链,诸多研究显示其氨基酸序列     

6q25区域内一个新基因MTLC的克隆及特性分析

目的：克隆6q25区域内喉癌相关新基因。方法：以表达序列标签为电子探针，在人类基因组数据库中进行电子杂交，钓出相就基因组DNA克隆后进行基因预测。根据获得的基因序列设计引物，逆转录－聚合酶链反应扩增新基因cDNA。结果：克隆了1个6q25区域内的新基因。该基因全长约21kb，含两个外显子，其cDNA序列长1006bp，编码蛋白包括235个氨基酸。其5’侧翼序列存在癌蛋白c-Myc的结合位点，将其命名为MTLC（c-Myc target from laryngeal cancer cells）基因。同源性分析表明该基因编码蛋白与小鼠MT－MC1蛋白同源性达78％。MTLC蛋白一级结构含有一个核定位信号结构域，亚细胞定位实验证实该基因主要在细胞核表达，Northern印迹分析表明MTLC基因在心肌、肝、肾、脑等多个组织有表达。结论：成功克隆了1个新基因MTLC，该基因可能作为c-Myc的靶基因以转录因子形式参与维持细胞正常生理功能。     

低密度脂蛋白诱导下调的新基因cDNA的克隆及组织表达

用改进的mRNA差异显示．PCR技术分析ECV304在低密度脂蛋白的诱导下表达水平明显差异的cDNA。获得-差异表达的265bp新EST；以该EST为探针，从人主动脉cDNA文库中筛选到一个1726bp的cDNA克隆，其748-1266bp之间的519个碱基构成一个完整的开放阅读框架，编码172个氨基酸组成的蛋白质。其羧基端94-172区间氨基酸序列中含有三个重复的C2H2型锌指蛋白基序CX2CX3FX5LX2HX3H，Northen Blot证实两组ECV304中均出现一条1．7kb的区带，LDL诱导后其表达降低了2．2倍。与差异显示-PCR的结果一致，该基因被定名为LRZFG。LRZFG是多组织表达的基因，其在动脉粥样硬化早期病理反应的作用有待于进一步研究。     

OPG与MBP融合蛋白在大肠杆菌中的表达

目的 克隆人骨保护素（OPG）成熟肽段编码区基因，并在大肠杆菌中表达。方法 采用RT－PCR法，扩增人OPG成熟肽段编码区cDNA，并克隆入原核表达载体pMAL-c2x中，转化BL21（DE3）PlysS大肠杆菌感受态细胞，经0.1mmol/L IPTG诱导后，收集菌体蛋白，进行SDS－PAGE及Western blot鉴定。结果 获得人OPG成熟肽段编码区cDNA，以构建的原核表达载体pMAL-OPG转化菌株后，可表达人OPG和麦芽糖结合蛋白（MBP）的融合蛋白，相对分子质量（M）为85000。表达产物的蛋白量约为菌体总蛋白的13％。Western blot表明，融合蛋白能与抗人OPG多克隆抗体特异性结合。结论 获得人OPG成熟肽全长cDNA，并在大肠杆菌中以OPC－MBP融合蛋白的形式表达。     

胃癌及其癌前病变相关新基因GP1的克隆

目的 比较胃癌、癌前病变和正常胃黏膜之间基因表达的差异,寻找与胃癌发生、发展相关的基因。方法 用荧光mRNA差异显示技术（FDD）分离差异表达的基因片段,进行PCR再扩增。将扩增的cDNA片段克隆后进行测序,测序结果提交GenBank,经BLAST软件检索进行同源性分析。差异条带经Northern杂交验证。应用SMART RACE（Rapid Amplication of cDNA Ends）技术扩增GP1的全长cDNA,并应用生物信息学技术预测该基因的生物学功能。结果 发现1个在胃癌及癌前病变组织中低表达的而且在GenBank数据库中未找到同源序列的cDNA片段,为新的基因片段,并获得GenBank登陆号CD454989。GP1的全长cDNA序列为1362bp,编码生物学功能未明的具有267氨基酸的蛋白质BAA91562.1。结论 发现了一个在胃癌、癌前病变及正常胃黏膜组织中差异表达的新基因,它可能参与了胃癌的发生、发展过程。     

卵黄萤荧光素再生酶基因的克隆与序列分析

目的克隆和分析荧光素再生酶基因（LRE）。方法通过GeneBank中已知的荧光素再生酶基因保守区段设计引物,利用5’RACE（rapid-amplification of cDNA ends）和3’RACE技术克隆了来自云南省西双版纳州的卵黄萤（Luciola ovalis）荧光素再生酶基因cDNA和全基因序列。通过GeneBank、National Center for Biotechnology Information和ProDom at the ExPASy Server软件和数据库进行序列分析。结果卵黄萤荧光素再生酶的cDNA序列和基因序列存在2个不同碱基位点,但是它们编码的荧光素再生酶是相同的。卵黄萤荧光素再生酶基因全长（从起始密码子到终止密码子）为1131bp,包含5个外显子4个内含子,其cDNA序列为1008bp,包含924bp的荧光素酶基因开放阅读框和84bp的3’UTR序列。卵黄萤荧光素酶基因的开放阅读框编码1个307个氨基酸的蛋白质。它与北美萤火虫（Photinus pyralis）荧光素再生酶在碱基序列和氨基酸序列上分别有61.8%和53.3%的相似性。结论成功地克隆了荧光素再生酶的cDNA和基因序列,为其在基因工程中的应用奠定了基础。     

Evidence for in vivo production of Humanin peptide,a neuroprotective factor against Alzheimer's disease-related insults

An unbiased functional screening with brain cDNA library from an Alzheimer's disease (AD) brain identified a novel 24-residue peptide Humanin (HN), which suppresses AD-related neurotoxicity. As the 1567-base cDNA containing the open reading frame (ORF) of HN is 99% identical to mitochondrial 16S ribosomal RNA as well as registered human mRNA, it was elusive whether HN is produced in vivo. Here, we raised anti-HN antibody and found that long cDNAs containing the ORF of HN (HN-ORF) produced the HN peptide in mammalian cells, dependent on the presence of full-length HN-ORF. Immunoblot analysis detected a 3-kDa protein with HN immunoreactivity in the testis and the colon in 3-week-old mice and in the testis in 12-week-old mice. HN immunoreactivity was also detected in an AD brain, but little in normal brains. This study suggests that HN peptide could be produced in vivo, and would provide a novel insight into the pathophysiology of AD.     

A human cDNA expression library in yeast enriched for open reading frames

We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.     

天使综合征致病基因UBE3a在大鼠神经元细胞中的克隆和表达检测

目的:调取并构建含有人UBE3a基因全长cDNA的真核表达载体pEGFP-UBE3a,采用磷酸钙转染方法将构建的重组质粒在神经元细胞中表达并观察EGFP-UBE3a蛋白的亚细胞定位。方法:采用PCR技术从人胎脑组织cDNA文库中扩增UBE3a基因的ORF片段并进行酶切构建pEGFP-UBE3a的真核表达重组质粒。用磷酸钙转染方法将重组质粒转染入体外培养的神经元细胞,分别在体外培养8天和14天时通过倒置荧光显微镜观察重组蛋白的表达和定位情况。结果:PCR扩增获得人胎脑组织cDNA文库中UBE3a cDNA全长2559 bp,构建真核表达重组质粒pEGFP-UBE3a成功,经DNA测序与Gen-Bank中的人UBE3a cDNA序列一致。经倒置荧光显微镜观察重组质粒的表达情况,结果显示转染重组质粒的细胞中有高水平的EGFP-UBE3a蛋白表达,且UBE3a在早期神经元的细胞质和细胞核中广泛表达,并弥散在轴突和树突,而在成熟神经元细胞核中的表达明显富集。结论:本实验成功构建了pEGFP-UBE3a真核表达质粒,EGFP-UBE3a蛋白在体外培养的神经元细胞中能够有效表达,且随着神经元的成熟逐步向核内富集,为进一步研究UBE3a基因在天使综合征发生发展中的作用奠定了基础。     

Sequence analysis of the 3'-end of feline calicivirus genome.

The nucleotide sequence of the 3'-end of the Japanese F4 strain of feline calicivirus (FCV) RNA was determined from a cloned cDNA of 3.5 kbp. We found three open reading frames (ORFs). The largest ORF encoded a 668-amino acid protein of 73,588 Da, which was presumably the capsid precursor protein of FCV and had significant amino acid sequence homology with the VP3 of picornaviruses. A small ORF at the extreme 3'-end was compared with that of the F9 strain of FCV, a vaccine strain originally from the U.S. Highly conserved amino acid sequences were shown, suggesting that this ORF might be functional and encode a putative 106-amino acid protein of 12,153 Da. The other ORF in the 5'-flanking region of the cDNA had consensus amino acid sequences conserved among the RNA-dependent RNA polymerases.     

人脑源性神经营养因子基因扩增及序列测定

目的：从基因组DNA获取编码脑源性神经营养因子基因,并对目的基因进行序列测定。方法：本研究直接从脑组织中提取人的基因组DNA,根据人的脑源性神经营养因子（BDNF）的cDNA序列,设计一对寡核苷酸引物,采用聚合酶链反应（RCR）,体外扩增编码人脑源性神经营养因子基因;扩增产物用自动分析仪进行序列测定。结果：从基因组DNA体扩增出脑源性神经营养因子基因,序列与献报道一致。结论：利用PCR直接从基因组获取目的基因,表明人脑神经营养因子基因为单一外显子,目的基因的扩增成功为进一步研究打下了基础。     

Molecular characterization of a<Emphasis Type="Italic"> Brugia malayi</Emphasis> transglutaminase

Prior studies have demonstrated that transglutaminase (TGase) from the human filarial parasite Brugia malayi is critical for the growth and development of the larval stages. In this report, we describe the cloning and partial characterization of a cDNA encoding the B. malayi TGase (BmTGase). Using RT-PCR and RACE-PCR, the cDNA was amplified from adult worm mRNA. BmTGase is 1,881 bp long and codes for a protein with a predicted molecular mass of 54 kDa. Amino acid sequence analysis of BmTGase revealed significant homology to the protein disulfide isomerase (PDI), particularly, to the PDI-related protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. The activity of recombinant B. malayi TGase enzyme (rBmTG) was found to be calcium-dependent and could be inhibited by EDTA. ELISA studies showed that approximately 88% of 48 sera from healthy Indian patients living in a bancroftian filariasis endemic area were reactive with rBmTG. In contrast, only 33% of sera from patients with clinical filariasis were reactive to rBmTG. Non-endemic sera were uniformly non-reactive. Additional studies are needed to elucidate the role, if any, of B. malayi TGase in protective immunity to filariasis.