抑癌基因PTEN载体构建及在LOVO细胞中的表达

目的:构建抑癌基因PTEN的表达载体并在LOVO细胞中表达。方法:从人外周血中提取总RNA,通过RT-PCR扩增出PTEN基因片段,将PTEN基因连入pLenti6/V5载体。测序鉴定后,经脂质体转染入LOVO细胞,对细胞株进行RT-PCR和Western blot检测。结果:DNA测序证实pLenti6/V5-PTEN表达载体为阳性克隆;该表达载体转染LOVO细胞后上调了PTEN mRNA和蛋白的表达。结论:成功构建了pLen-ti6/V5-PTEN表达载体,为进一步研究PTEN在LOVO细胞中的功能奠定了基础。     

链霉菌查尔酮合成酶基因克隆及原核表达载体构建

根据Genbank数据库已知的链霉菌的查尔酮合成酶基因的保守区设计chs基因特异简并引物,土壤总DNA为模板,利用PCR技术扩增得到该1条chs基因编码区,通过TA克隆、测序和同源比对及进化分析表明：该基因为放线菌来源chs基因．分别在该基因5＇末端和3’末端分别引入的限制酶NcoI和EcoRI酶切位点,利用上述2种限制酶分别酶切导入到psimple—T／chs载体和原核表达pET32a,凝胶回收目的片段后,将二者连接并转化大肠杆菌感受态细胞,转化子经菌液PCR筛选、双酶切鉴定后,3730测序结果表明,该基因全长编码区为1089bp,推测该基因编码全长为362个氨基酸残基,等电点（PI）为5．41、分子量为3965道尔顿含有cHs保守功能区的酸性蛋白质．分析表明,该基因与Streptomyceslividans来源的查尔酮合成酶RppA基因核苷酸相似性高达93％,氨基酸序列相似性高达87．70％．测序结果表明,该基因已经成功插入到pET32a载体中．     

Spatio-temporal expression of the pathway-specific regulatory gene redD in S. coelicolor

INTRODUCTION Streptomycetes are gram-positive soil bacteriarenowned for their ability to produce a large numberof different secondary metabolites. Their life cycleinvolves complex morphological differentiation,which is believed to be closely co-ordinated with geneexpression that promotes a variety of physiologicaland structural changes including the onset of antibi-otic biosynthesis. S. coelicolor has been extensivelystudied as a model system with the aid of genetic andmolecular technique…     

SEREX技术筛选EID3及其表达的实验研究

[目的]新的大肠癌相关性抗原EID3的基因克隆及其诊断价值研究.[方法]利用大肠癌病人体内血清中所含的对肿瘤抗原产生的特异性抗体筛选睾丸组织cDNA噬菌体表达文库和大肠癌组织cDNA噬菌体表达文库(SEREX),并用RT-PCR技术研究EID3 mRNA在正常组织和大肠癌传代细胞表达.[结果]睾丸组织cDNA噬菌体表达文库筛选得到了可以诱导大肠癌病人抗体免疫应答的新抗原EID3基因(Gen-bank NM_001008394.1).它们定位于染色体19q13.2,EID3含1个外显子.通过RT-PCR分析发现,EID3基因在43例大肠癌传代细胞株中,39例阳性,阳性率为90.7%.在正常组织中,除睾丸组织外不表达或有极低水平转录.[结论]EID3 mRNA表达检测用于诊断大肠癌,可能具有高特异性和高敏感性的特点.EID3蛋白被首次发现在大肠癌病人中能够诱导机体的抗体免疫应答,为一个新的大肠癌相关性抗原分子.其功能可能与抑制细胞的恶性增殖相关,并可进一步研究其用于治疗和诊断大肠癌的可行性.     

食道癌和胃癌组织中MDM2基因蛋白表达的研究

为探讨MDM2基因蛋白在胃、食道癌中表达的特点。我们利用SABC免疫组织化学方法,检测了69例食道癌和胃癌标本。结果表明:MDM2总阳性率为56.52％,在食道癌中阳性率为63.47％,胃癌中阳性率为46.42％;在高分化癌组织中阳性率为77.78％;低分化癌阳性率为29.41％;两者差异有显著性(P<0.05)。文中对MDM2基因在以上两种癌组织中表达的意义进行了讨论。     

人胰岛素样生长因子Ⅰ型在E.coli和家蚕中的表达

将hIGF-I基因克隆进原核表达载体pET-28a( ),在E.coli中进行了融合表达,West-ern blotting显示在26 kD附近有一条特异条带。将hIGF-I基因克隆进pBacPAK-8,获得了杆状病毒转移载体pBacPAK-8-IGF-I,在脂质体的介导下,与线性化的家蚕杆状病毒共转染家蚕培养细胞Bm-N,经空斑筛选,PCR检测,获得了重组病毒Bm-Bac-hIGF-Ⅰ。SDS-PAGE检测表明,在感染重组病毒后,家蚕幼虫血淋巴中可以检测到一条分子量约为7.5 kD的特异性条带,ELISA检测表达量达4.51μg/mL蚕血淋巴。     

Sequence analysis and expression pattern of MGTA1 gene in rice blast pathogen Magnaporthe grisea

INTRODUCTION Fungi cause most plant diseases of all groups of microbes. During their infection cycles, fungal pathogens must undergo two key processes: first, penetration through cuticles into host plant cells; second, colonization in host cells utilizing nutrients from their hosts. To penetrate host cells, fungi de-velop a series of specialized infection structures such as appressorium, penetration peg, and infection hypha. The appressorium-mediated penetration is a process typical of s…     

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of <Emphasis Type="Italic">Bombyx mori</Emphasis> fibroin light chain gene in vitro

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmomiR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L. To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pribmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected by pcDNA3.0 [ie1-egfp-pri-bmomiR-0001-SV40] or pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with pGL3.0 [A3-luc-Fib-L-3′UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro.     

RRM1 gene expression in peripheral blood is predictive of shorter survival in Chinese patients with advanced non-small-cell lung cancer treated by gemcitabine and platinum

Objective  

To evaluate the predictive values of gene expressions of ribonucleotide reductase M1 (RRM1) and breast cancer susceptibility gene 1 (BRCA1) in peripheral blood from Chinese patients with non-small-cell lung cancer (NSCLC) treated with gemcitabine plus platinum.