热休克蛋白(HSPs)基因家族在涡虫中的研究进展

热休克蛋白(HSPs)广泛存在于从细菌到人类的各种有机体中,在生物生命活动中具有重要生理功能,如作为分子伴侣参与细胞增殖和分化以及免疫应答等;同时某些HSPs表达水平的高低可作为衡量环境污染或者人类恶性肿瘤疗效和预后的重要指标.涡虫是扁形动物门的代表动物,因其在动物系统演化中的特殊地位和极强的再生能力而作为研究发育和再生的模式动物之一.概述了HSPs基因家族成员在涡虫抗逆和再生研究中的主要成果和最新进展,并对目前存在的问题及未来的发展方向进行了总结和展望.以期为揭示HSPs在高等动物中的作用机制提供参考.     

脂肪酶产生菌的筛选及基因的克隆表达

目的筛选出脂肪酶活性较高菌株,通过脂肪酶基因克隆技术获得高表达的脂肪酶,根据酶活曲线确定该酶的最适温度和最适pH.方法采用透明圈法,以三丁酸甘油酯为底物筛选脂肪酶活性较高菌株;通过PCR扩增获得其脂肪酶基因Pseudomonas peli(PP)序列,构建pET-28 a表达载体,并在大肠杆菌Escherichia coli BL21(DE3)中进行异源表达;通过Ni柱将脂肪酶纯化,并根据酶活曲线确定该酶的最适温度和最适pH.结果筛选到一株脂肪酶活性较高的菌株,16S rRNA基因序列比对结果初步鉴定为Pseudomonas peli.PCR扩增得到的基因片段长度为801 bp,其序列与Pseudomonas peli中的脂肪酶基因序列相似性达到100%,PP基因在大肠杆菌中异源表达蛋白的分子量约29 KD,该蛋白在55℃、pH 7.0时活性最高.结论通过基因克隆表达后得到的脂肪酶Ni柱纯化后纯度达95%以上,且耐高温,为脂肪酶工业化应用奠定了基础.     

类硫氧还蛋白hTRXLⅠ cDNA的克隆和原核表达

从人18周胎脑中抽提mRNA,建立cDNA文库,通过大规模测序克隆出一条人类基因,该基因全长1644bp,开放阅读框1146bp,编码一个含372个氨基酸的蛋白,预测分子质量约为46.8ku.经与蛋白数据库比较,发现具有人硫氧还蛋白结构,命名为人的类硫氧还蛋白基因Ⅰ(human Thioredoxin like geneⅠ,hTRXLⅠ).多组织膜Northern blot结果显示在心脏、脑、胎盘、肝、骨骼肌、肾、胰腺等各个组织中均有表达,肺中表达不明显.通过Unigene染色体定位,将hTRXLⅠ定位于11q11,把hTRXLⅠ的读框克隆入经改造的PBV220质粒中,在E.coli中诱导后获得表达.     

Plant polar growth in tobacco disturbed by y-tubulin gene silencing

To further understand the functions of y-tubulin in plant cells, we conducted a study in which the y-tubulin gene was down-regulated in tobacco plants (obtained by the Agrobacterium-mediated method). This involved transforming the target fragments, in which the sense and antisense partial y-tubulin cDNA fragments were ligated together, into Nicotiana tabacum var. Samsun NN. The y-tubulin down-regulated transformants developed multiple meristems or branches with trumpet-shaped leaves; their root generation also appeared abnormal, with the taproots undeveloped, whereas lateral roots were developed. In addition, the content of indole-3-acetic acid (IAA) and expression of polarity transportation vector PGPI were aberrant. These results suggest that y-tubulin gene silencing disturbed the polar growth of tobacco plants, and that this phenomenon was probably correlated with the IAA content and the polar transpor-tation process.     

鸡腺病毒内蒙古分离株六邻体蛋白基因片段的合成和分子克隆

根据鸡１０型腺病毒以及人２、５、４０、４１型腺病毒、牛３型、鼠１型腺病毒六邻体蛋白基因序列，选择保守区，设计和合成一对引物，以鸡腺病毒内蒙古分离株基因组ＤＮＡ为模板，进行聚合酶链反应（ＰＣＲ）扩增得到预期大小的０．５５ｋｂＤＮＡ片段．将此ＤＮＡ片段克隆于ｐＵＣ１９的ＳｍａＩ位点，筛选重组质粒，进行限制酶切分析和ＰＣＲ检测，得到含有六邻体蛋白基因片段的重组质粒，为进一步开展此病毒分子生物学研究和分子生物学诊断技术的建立创造了条件     

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.     

Cationic polymeric nanoformulation: Recent advances in material design for CRISPR/Cas9 gene therapy

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat associated proteins 9) gene editing platform is a promising therapeutic tool for genetic disorders, due to its ability to manipulate the pathogenic gene in genomic level and to easily target specific gene by manipulating single-guide RNA. However, its successful delivery remains a challenge. Up to now, great efforts have been made to explore an effective strategy for CRISPR/Cas9 delivery. But among those delivery methods, physical methods are mainly operated on cultured cells thus limited to laboratorial use; viral vectors are hindered by fetal immunogenic and carcinogenic effects thus dubious in clinical application. Therefore, cationic polymeric vectors, with the ability to interact with CRISPR/Cas9 system to form a nanoformulation as a non-viral approach, are attracting increasing attentions, due to advantages such as well protection of cargos, less limitation in payload size, low immunogenicity or carcinogenicity, potential modifications for further functions, and ease in mass production. In this review, the recent discoveries on polymeric vectors utilized in delivery of CRISPR/Cas9 system will be summarized. With emphasis on advanced features of those polymeric vectors or their nanoformulations to meet the demands of different CRISPR/Cas9 delivery forms (plasmid, mRNA or protein), the detailed illustrations on their disease treatment applications, such as cancer, diabetes or antibiotic-resistant infections, will also be reviewed.     

人类新基因克隆

本文概述了人类基因组计划及人类基因组研究的新进展,重点阐述了人类新基因克隆的策略与现状、问题与对策,同时就新基因克隆过程中的技术难点作了概括性的归纳,并就其突破分别提出可行的建设性意见,对从事这方面研究的科研人员具有重要的指导作用。