带信号肽DsbA-胸腺素α1融合蛋白的表达及其分离纯化

以构建好的人胸腺素α1基因工程菌为研究材料,研究影响DsbA-胸腺素α1融合蛋白分泌效率的因素,优化信号肽酶表达得到阻遏的培养条件,获得含带信号肽DsbA-胸腺素α1融合蛋白的菌体,破碎细胞后利用疏水层析与Ni2 螯合亲和层析进行目标蛋白分离纯化,获得较高纯度底物蛋白(信号肽DsbA-胸腺素α1融合蛋白,其分子量约为34 kD),为进一步研究及分离纯化DsbA信号肽酶创造条件.     

LC-1 ScFv偶联GFP基因的构建及表达

从具有高效蛋白表达及活力的抗肺癌杂交瘤单克隆细胞株（LC-1）抽提总RNA，反转录成cDNA。根据肿瘤单链抗体（ScFv）基因设计引物，用多聚酶链式反应（PCR）克隆出抗体的重链和轻链基因，并通过重叠延伸PCR法将重链基因与修饰后的轻链基因连接为单链抗体基因。改造后的ScFv与绿色荧光蛋白（GFP）基因连接，构建表达质粒ProGFP-ScFv，随后转化大肠杆菌JM109，用诱导剂IPTG进行诱导表达。表达产物采用Ni-NTA树脂进行纯化，进行SDS-PAGE电泳分析其纯度。酶联免疫检测（ELISA）表明该蛋白已具有抗体活性。395 nm激光激发显示绿色荧光，表明目的基因在原核生物中存在表达。     

人胸腺素α1基因工程菌的构建与表达

胸腺素α1(Thymosin alpha 1,Tα1)是一种针对T淋巴细胞的免疫增强剂,临床用途广泛.为大量制备Tα1,按大肠杆菌惯用密码子合成编码Tα1的基因,克隆于pUCmT,转化E.coli JF1125,经测序证明序列正确后,克隆入pET39(b)的BspT I和BamH I位点,成功地构建出包含信号肽,DsbA,连接肽,6个His,EKsite(Asp-Asp-Asp-Asp-Lys),胸腺素α1的表达载体,转化E.coliBL21(DE3),获得胸腺素α1基因工程菌.SDS-PAGE分析表明,经0.1 mmol/L IPTG和30℃诱导8 h,在大肠杆菌周质中大量表达表观分子量31 kD左右的融合蛋白.为进一步获得大量可供实验研究和临床应用的重组胸腺素α1创造条件.     

骨形态发生蛋白-2基因工程菌的构建及可溶性研究

骨形态发生蛋白是一类调节骨组织发育的生长因子,其中骨形态发生蛋白-2诱导成骨活性最强,在骨组织工程研究中最具研究意义.为获得能高效表达溶解性高的人骨形成蛋白-2(BMP-2)的基因工程菌,用PCR方法扩增得到BMP-2的基因序列,直接将PCR产物连接到胞内融合表达型T载体质粒pMT-L上,构建包括麦芽糖结合蛋白(MBP)、连接肽、6个His、EKsite(Asp-Asp-Asp-Asp-Lys)和BMP-2的表达载体,转化E.coli DH5α,经抗性筛选和菌落PCR鉴定,抽提阳性克隆质粒转化表达宿主E.coli BL21(DE3),成功构建可在大肠杆菌细胞质内表达MBP-BMP-2融合蛋白的基因工程菌.工程菌经0.1mmol/L IPTG诱导后,可获得表观分子量约为55kD的以可溶形式表达的BMP-2融合蛋白.     

人参3-O-UGT1基因RNAi表达载体工程菌的构建

人参3-O-UGT1基因(Pg3-O-UGT1)是控制人参中二醇型单体皂苷合成的关键酶基因,而二醇型皂苷和三醇型皂苷具有相反的药理活性。本文利用RT-PCR技术和酶切连接技术成功构建人参3-O-UGT1基因RNAi(RNA interference)植物表达载体,通过热转化法将重组表达载体转化进发根农杆菌A4中,得到含有人参3-O-UGT1基因RNAi植物表达载体的工程菌,为诱导人参外植体转化和人参皂苷合成机理奠定了基础。     

酵母多基因表达载体在纤维素生物转化中应用

构建含酿酒酵母组成型强启动子PGK、G418抗性基因及rDNA片段的整合型载体pScIKP,利用rDNA多个同源重组位点,将外源基因以多拷贝整合到酵母染色体上,无需诱导即可持续表达;利用载体位于表达盒两端同尾酶,可插入多个基因表达盒,实现多基因稳定共表达.为检验共表达情况,反转录从绿色木霉中获得纤维素酶基因eg3和cbh2, 克隆并转化获得重组酵母菌株S.cerevisiae-ec. 该重组酵母能降解羧甲基纤维素形成水解圈;用羧甲基纤维素还原糖法和滤纸酶活力法测定酶活力,其最适温度和最适pH值与所表达单酶相似,表明共表达未影响两种酶的生物学特性;且双酶具有协同作用,能更有效降解非结晶纤维素.pScIKP载体能成功用于多个外源基因共表达和产物协同作用的研究,为构建能直接降解纤维素的酿酒酵母菌株,实现纤维素可再生能源的生物利用奠定了基础.     

Surface Modification of Biomimetic PLGA- （ASP-PEG） Matrix with RGD-Containing Peptide： a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.     

一个改进的LR（1）分析表及其构造算法

LR（1）分析表是LR（1）分析器的核心。改进了传统的LR（1）分析表,提出了新的构造算法。该算法利用LR（1）基本集代替LR（1）项集,对于归约状态直接标注归约转移后的状态编号。该分析表不含GOTO表,基于它的LR（1）语法分析过程一般不需要后入先出栈的辅助,文中的一个实例说明了该分析表的有效性。     

基于网格搜索和支持向量机的灰熔点预测

为了预测混煤的灰熔点,采用支持向量机建立煤灰软化温度模型,模型将煤的灰成分作为输入量,煤的软化温度作为输出量,利用网格搜索寻优方法对支持向量机(SVM)模型的参数进行了优化,在设定的不同精度下分别获得模型的最优参数,利用优化后的模型对单煤和混煤的灰熔点进行了预测,并将不同精度下的预测结果与实验结果进行对比.煤灰软化温度模型设定精度为0.01时,单煤样本预测相对误差最小,其最大相对误差和平均相对误差分别为3.00%和0.48%;运用此模型对混煤预测的最大相对误差和平均相对误差分别为1.74%和0.62%.预测结果表明,经网格搜索优化后的支持向量机模型对煤灰熔点预测较精确.     

Synthesis and characterization of soluble and meltable poly(aminopropyl/phenylsilsesquioxane)

The hydrolytic co-condensation of hydrophobic phenyltriethoxysilane (PTES) and hydrophilic γ-aminopropyltriethoxysilane (APS) was investigated in toluene and water by using hydrochloric acid (HCl) catalyst. A soluble and meltable poly(aminopropyl/phenylsilsesquioxane) (PAPSQ) was formed by controlling HCl amounts, APS/PTES molar ratios, water/silane molar ratios (Rw/si), organic co-solvents and re-equilibration steps as well. The compositions of PAPSQ bearing aminopropyl and phenyl groups with the capping of trimethylsilyl group were confirmed by element analysis, fourier-transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) and thermogravimetric analysis (TGA). Differential scanning calorimetry (DSC) was employed to characterize the melting behavior of the product. PAPSQ can be easily employed as functional molecular building blocks for the synthesis of diverse and novel inorganic-organic materials.