High expression of human augmenter of liver regeneration in E. coli

Heat-stable hepatocyte stimulatory activity has been described in the liver of weanling rats and pigs. This growth factor is called hepatocyte stimulator substance (HSS)[1]. Hagiya et al[2]reported the complete amino acid sequence of a 30KDa band from their purified product of rat liver and the cloning and sequence analysis of its cDNA. They called it augmenter of liver regeneration (ALR). Our previous study[3]demonstrated that human ALR had been cloned and sequenced. To further study the bioactivity of human ALR (hALR), we constructed a highly expressed vector pBV-hALR in E. coli and about 20% of somatic protein of rhALR was expressed.     

靶向XT-1基因shRNA真核表达质粒载体的构建及筛选

目的构建靶向木糖转移酶-1（XT-1）的短发卡状小干扰RNA（shRNA）质粒表达载体;为寻找脊髓损伤治疗的新靶点提供工具。方法设计4个小分子短发卡状RNA（siRNA）序列,体外合成DNA模板引物,与Pgenesil-1质粒构建成编码shRNAR的表达载体,进行酶切和测序,再转染体外培养的星形胶质细胞,筛选靶序列。结果构建的质粒表达载体完全符合设计要求,转染成功的细胞在荧光显微镜下显示绿色荧光,G418可以筛选出阳性克隆。结论成功构建了编码XT-1-shRNA的质粒表达载体;该载体为寻找脊髓损伤治疗的新靶点奠定了基础。     

重组耻垢分枝杆菌Msmc^2-CFP10-ESAT6的构建

目的构建能表达结核分枝杆菌早期分泌蛋白CFP10-ESAT6融合蛋白的重组耻垢分枝杆菌。方法采用基因拼接（Gene SOEing）法体外扩增结核杆菌1hp-ESAT6融合基因,插入大肠埃希菌-分枝杆菌穿梭表达质粒pB-CG3000,构建重组穿梭表达质粒pBCG3000-CFP10-ESAT6,电转化耻垢分枝杆菌mc2155,构建耻垢疫苗株Msmc2155-CFP10-ESAT6,热诱导表达CFP10-ESAT6融合蛋白,Western blot分析其免疫原性。结果经PCR、酶切及测序鉴定,证实成功构建重组质粒pBCG3000-CFP10-ESAT6,并在耻垢分枝杆菌中经热诱导表达出分子质量单位约为22 ku的CFP10-ESAT6蛋白,该蛋白可被结核病患者血清、鼠抗ESAT6血清和鼠抗CFP10血清识别。结论结核分枝杆菌lhp-ESAT6融合基因在耻垢分枝杆菌中成功表达。     

弓形虫表面抗原P30、P22与霍乱毒素A2／B复合基因真核表达质粒的构建

目的选择弓形虫 RH 株主要表面抗原 P30、P22的有效基因片段和霍乱毒素 A_2/B 亚基共同构建在同一真核表达载体 pcDNA3.1(-)上,并保证其连接方向和开放读码框的正确。方法用PCR 技术分别从弓形虫 RH 株基因组 DNA 和 pUAB024-CTXA_2/B 质粒中扩增编码 P30、P22基因片段和 CTXA_2/B,定向重组入 pUC18克隆载体,然后酶切释放 P30-P22-CTXA_2/B 复合基因片段,亚克隆入pcDNA3.1(-)真核表达载体,再经含氨苄青霉素的 LB 培养基筛选、酶切及测序鉴定。结果酶切产物经电泳显示条带清晰,P30、P22和 CTXA_2/B 基因片段的泳动位置分别在786bp、492bp、512bp 的位置,与预计结果一致;测序结果表明插入的基因片段方向及序列均正确。结论成功地构建了真核表达重组质粒 pcDNA3.1-P30-P22-CTXA_2/B,保证了三个基因连接方向,序列及开放读码框的正确,为下一步融合蛋白 P30-P22-CTXA_2/B 在哺乳动物细胞中的表达及动物实验奠定了基础。     

含弓形虫复合抗原基因的真核表达质粒在哺乳动物细胞中的表达

目的研究弓形虫复合抗原真核表达质粒pcDNA3.1-P30-P22-CTXA2/B在哺乳动物细胞中的表达情况。方法利用脂质体介导的转染技术,将真核表达质粒pcDNA3.1-P30-P22-CTXA2/B和空载体pcDNA3.1分别转染He-la细胞,400μg/mlG418加压筛选和200μg/mlG418维持筛选,获得稳定转染的Hela细胞。采用SDS-PAGE和West-ern-blot方法对复合基因P30-P22-CTXA2/B的表达产物进行鉴定。结果SDS-PAGE结果显示,重组质粒转染Hela细胞后的表达产物分子质量单位为64ku,Western-blot显示此蛋白条带能被抗P30抗体识别。结论构建的真核表达质粒pcDNA3.1-P30-P22-CTXA2/B能在哺乳动物细胞中成功表达插入基因所编码的融合蛋白,为进一步动物实验提供了实验依据。     

人内皮抑素小环载体在真核细胞中的生物活性

目的通过对人内皮抑素小环载体在真核细胞中的表达和生物学效应的研究,探讨小环载体作为生物治疗载体的可行性。方法将mc-hES、pcDNA-hES分别转染人肝癌细胞HepG2后,通过ELISA、RT-PCR和Westernblot观察hES表达。MTT法检测其对人脐静脉内皮细胞（HUVEC）的增殖抑制作用。结果 mc-hES和pcDNA-hES均能表达hES,mc-hES能明显抑制HUVEC的生长,而对HepG2无明显作用（P〈0.05）。在相同条件下,小环较常规质粒在体外能快速介导转基因的表达,且较传统质粒高6～8.3倍。结论 mc-hES较普通质粒pcDNA-hES在肝癌细胞中能高效表达转基因,为小环载体进一步研究提供了很好的实验基础。     

High resolution cell lineage tracing reveals developmental variability in leech

Knowing the normal patterns of embryonic cell proliferation, migration, and differentiation is a cornerstone for understanding development. Yet for most species, the precision with which embryonic cell lineages can be determined is limited by technical considerations (the large numbers of cells, extended developmental times, opacity of the embryos), and these are exacerbated by the inherent variability of the lineages themselves. Here, we present an improved method of cell lineage tracing in the leech Helobdella, driving the expression of a nuclearly localized histone H2B:GFP (green fluorescent protein) fusion protein in selected lineages by microinjection of a plasmid vector. This construct generates a long lasting and minimally mosaic signal with single cell resolution, and does not disrupt the development of most lineages tested. We have validated this technique by elucidating details of cell lineages contributing to segmental and prostomial tissues that could not be observed with standard dextran lineage tracers. Developmental Dynamics 238:3139–3151, 2009. © 2009 Wiley‐Liss, Inc.     

Genetic engineering for haemophilia A

At first sight, haemophilia A would appear to be an ideal candidate for treatment by gene therapy. There is a single gene defect; cells in different parts of the body, but especially the liver, produce Factor VIII, and only 5% of normal levels of Factor VIII are necessary to prevent the serious symptoms of bleeding. This review attempts to outline the status of gene therapy at present and efforts that have been made to overcome the difficulties and remaining problems that require solving. Undoubtedly, success will be achieved, but it is likely that considerably more work will be necessary before experimental models can be introduced into the clinic with any likelihood of success. The most successful results in animals that may have clinical application were from introducing the Factor VIII gene to newborn animals before antibodies are produced, presumably inducing a state of tolerance.     

泛素结合酶E2-EPF高表达质粒的构建及对绒癌细胞JEG-3生长功能的初步探讨

目的：构建泛素结合酶E2-EPF高表达质粒,初步探讨E2-EPF在绒癌细胞增殖中的作用。方法：基因克隆技术构建pcDNA（3.1＋）-E2-EPF高表达质粒,通过瞬时转染将质粒导入绒癌细胞JEG-3中,使E2-EPF高表达;流式细胞仪检测及细胞增殖实验初步研究E2-EPF高表达对绒癌细胞生长速率的影响。结果：E2-EPF高表达质粒构建成功,瞬时转染后细胞E2-EPFmRNA升高约8.4倍,JEG-3细胞的生长速率显著高于对照组（P〈0.05）,处于G2-M期和S期细胞显著增加。结论：E2-EPF参与细胞的生长调控,促进E2-EPF表达可以加速绒癌细胞JEG-3的生长速率。