Optimization of the production of Aspergillus niger α-glucosidase expressed in Pichia pastoris

The α-glucosidase (AGL) from Aspergillus niger has been applied to produce isomaltooligosaccharides. In the present study, various factors which affect the yield of recombinant AGL, produced by engineered Pichia pastoris, were investigated. The expression level reached 5.5 U ml^?1 in bioreactor after optimization of parameters of initial induction cell density, induction temperature and methanol concentration. In addition, it was found that coexpression of protein disulfide isomerase (PDI) inhibited the growth of the engineered P. pastoris strains and had an adverse effect on the production of AGL, while codon optimization of native A. niger α-glucosidase encoding gene (aglu) resulted in a significant enhancement of enzyme production, which reached 10.1 U ml^?1. We believe that yield of AGL is increased by codon optimization as a result of enhanced translation efficiency as well as more stable mRNA secondary structure. In contrast, PDI coexpression under the control of alcohol oxidase promoter (PAOX1) seems to be less efficient in helping disulfide bond formation in AGL while probably induce unfolded protein response, which further leads to cell apoptosis and increased protein degradation.     

Proteomic Analysis for the Identification of Proteins Related to Methotrexate Resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 μmol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.     

Kisspeptins在孕早期血浆和胎盘组织中的表达及其意义

目的:研究kisspeptins在孕早期血浆和胎盘组织中的表达,拟探讨其来源及其在妊娠中意义.方法:采用酶联免疫吸附法(ELISA),检测40例孕早期妇女和35例正常未妊娠妇女血浆kisspeptins的表达水平,且分析其与hCG的相关性;采用免疫组织化学方法进行其组织学定位.结果:(1)kisspeptins在妊娠组...     

小鼠外胎盘锥细胞与层粘连蛋白相互作用机制的研究

本文用LN及含其活性位点序列的合成肽段cYIGSR和RGDS对小鼠EPC细胞与LN的相互作用机制进行研究。结果表明:合成肽段cYIGSR和RGDS能促进EPC粘附并具协同效应。cYIGSR还能促进EPC扩展与次生TGCs迁移。LNA链上RGD和B_1链上YIGSR两个活性位点协同地参与了LN对EPC的粘附、扩展以及次生TGCs的迁移的促进作用。cY R合用不能完全竞争性抑制EPC与LN的结合,说明还有其他作用位点存在。     

噬菌体抗体文库的构建及人源抗HIV-1 gp 160抗体的筛选

构建人源噬菌体抗体文库如下：ＨＩＶ感染者脾细胞中提取ｍＲＮＡ,经反转录再以人ＩｇＧ重链Ｆｄ两端及轻链“通用”引物分别扩增Ｆａｂ基因片段,依次插入到噬菌粒载体ｐＣｏｍｂ３中,电转化大肠杆菌ＸＬ１－Ｂｌｕｅ,经辅助噬菌体救助,重组噬菌体得以溶源裂解,Ｆａｂ表达于噬菌体包膜蛋白Ⅲ的Ｎ端．此噬菌体抗体库的容量为５×１０５．筛选抗ＨＩＶ－１同时又具有能被抗独特型抗体“ＩＦ７”所识别的独特型的阳性抗体：以重组包膜糖蛋白ｇｐ１６０及ｇｐ４１多肽对噬菌体抗体文库进行三次淘选,使抗ｇｐ１６０的特异性抗体得到１００倍的富集．然后通过直接ＥＬＩＳＡ和竞争性ＥＬＩＳＡ实验筛选出两株结合性较好的特异性抗ｇｐ１６０抗体－３Ｂ株与１Ｄ株．直接ＥＬＩＳＡ实验表明这两株克隆均能被“１Ｆ７”所识别,为抗独特型多肽的筛选奠定了基础．     

蛇毒类凝血酶鼠单克隆抗体的制备

采用杂交瘤技术,获得了４株稳定分泌抗蛇毒类凝血酶的单克隆抗体杂交瘤细胞株,均属ＩｇＧ１ｋ链,４株杂交瘤细胞培养上清液效价为 ４ × １０－１～４ × １０－２,腹水效价为 ４ × １０－１～３．２ ×１０－５。     

高脂血症兔球结膜微循环的改变

本实验用胆固醇粉喂家兔,造成实验性高脂血症,活体观察球结膜微循环的形态、流态及血管周围状态等１５项指标,并用球结膜微循环综合定量评价方法计算了综合积分值。处死后取球结膜进行组织学检查。结果表明：高脂血症家兔球结膜微循环有明显改变,主要为微血管形态异常、血流减慢、红细胞聚集、白微栓、静脉壁上有白色斑块等改变。球结膜微循环综合积分值明显增加,高于实验前。球结膜组织学检查发现上皮层下有泡沫细胞、血管壁增厚、有空泡、静脉内有血栓。虹膜睫状体内除有多数泡沫细胞外,还有胆固醇的菱形结晶。上述改变表明,高脂血症兔球结膜微循环有明显改变。血液有高凝、高聚倾向。黑茶中的茶色素有抗凝、抑制血小板聚集、促进纤溶等作用,口服黑茶可改善高脂血症兔球结膜微循环障碍。     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

4C-克隆筛选过程中的质量控制

环形染色体构象俘获(Circular chromosome conformation capture,4C)是一种高通量研究细胞染色体相互作用、空间构象的技术。文章通过模拟的4C样品,优化反向PCR条件,建立了高效、特异的扩增方法,对4C克隆筛选等后续步骤进行严格的质量控制并对该方法的可行性进行了实例验证。该4C-克隆筛选方法作为4C方法的质量控制标准,具有重要的指导和监测作用。     

维甲酸诱导的脊柱裂小鼠的脊髓全基因组表达谱分析(英文)

关于维甲酸胚胎病理学的研究很多,维甲酸受体在器官发生、发育及神经管闭合过程中发挥重要作用。但维甲酸影响这些过程的机制还不清楚。在本研究中,我们发现,小鼠怀孕8天时,给予母体连续3次维甲酸灌胃,将导致胎儿脊柱裂,发生率为96.77%。本研究应用微阵列技术,在维甲酸诱导的脊柱裂小鼠胎儿的脊髓组织中发现了134个差异表达在1.5倍以上的基因。基因富集分析显示,母亲暴露于维甲酸导致的胎儿脊柱裂,与促凋亡和抗凋亡、细胞增殖、迁徙、细胞骨架成分以及细胞或局部粘附等基因功能簇相关,提示这些细胞成分和生物学的功能缺陷促使脊柱发育异常。我们的研究提供了脊柱裂的全基因组基因表达模式,有助于理解神经管缺陷的病因和病理学。