pEgr-ssEndostatin的构建及其在体外的辐射诱导表达

为探讨pEgr-ssEndostatin重组质粒转染B16细胞后接受不同辐射剂量以及接受同一剂量不同时程endostatin表达的规律。采用RT-PCR方法从鼠肝脏中扩增出Endostatin cDNA,连入信号肽,构建了pEgr-ssEndostatin重组质粒,用脂质体介导的转染法转染小鼠B16细胞,用ELISA方法检测B16细胞上清中endostatin的表达水平。结果表明的分泌型Endostatin序列与报道完全一致,Egr-1启动子和Endostatin cDNA正确插入表达载体pcDNA3．1;pEfr-ssEndostatin具有辐射诱导表达特性。提示小鼠Endostatin基因成功地被克隆和表达,Egr-1启动子具有辐射激活和诱导下游基因表达增强的功能。     

Effects of nutrients on α-amyiase production and plasmid stability in fed-batch culture of recombinant bacteria

Escherichia coli harbouring a recombinant plasmid containing the α-amylase gene (HB101/pHI301) and its derivative which can stably maintain the plasmid (HB101/pHI301A) were cultivated in a mini-jar fermenter. By controlling dissolved oxygen (DO) at a concentration of 2–3 μg/l and supplementing with suitable amounts of nutrients, the organisms could grow to a concentration (up to 40 g dry cell l^?1) in a semi-synthetic casamino acid medium. During culture the plasmid was maintained stably in both strains. In a synthetic medium, both micro-organisms grew less well and plasmid-free segregants were observed with HB101/pHI301 during the fed-batch culture. On the other hand, plasmid pHI301A was stably maintained throughout the fed-batch culture of HB101/pHI301A. Growth inhibitors, which may be metabolic products, accumulated in the medium and reduced bacterial growth.     

Effects of phosphoglycerate kinase overproduction in Saccharomyces cerevisiae on the physiology and plasmid stability.

In this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyces cerevisiae containing the PGK1 gene on an episomal plasmid are described. This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10-15% of the total soluble protein. Strains containing the plasmid and the host strain were grown in non-selective batch cultures and continuous culture, under different growth conditions. Plasmid-containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12%. This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose-limited conditions. During respiro-fermentative growth, PGK-overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain. The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. The results are consistent with previously reported data on the physiology and gene stability of PGK-overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.     

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.     

DNA分子杂交技术在基因工程人白细胞介素-2质量检测中的应用

将携带有人白细胞介素-2基因的工程菌(PBV~(220)/ZL-2),发酵培养后经一系列提纯可获得基因工程人白细胞介素-2(rIL-2)的纯品。采用分子杂交技术,用~(32)P标记PBV_(220)大肠杆菌全DNA为探针,检测rlL-2纯品,没有检测出残余DNA,完全符合WHO和“中国人用重组DNA制品质量控制要点”所规定的残余DNA含量在100Pg/剂以下的要求。     

Factors affecting homologous overexpression of the Saccharomyces cerevisiae lanosterol 14 alpha-demethylase gene.

The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S. cerevisiae using promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values. With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.     

注射用重组葡激酶的中试研究

在已获得了高效表达重组葡激酶工程菌在基础上建立了快速、高效的注射用重组葡激酶中度生产规程。工程菌经机械破碎和离心后，上清液经硫酸铵分级沉淀，一步凝胶过滤和一步阴离子交换即可获得高纯度、高活性的半成品，加入稳定剂后过滤除菌，分装，冻干即得成品，蛋白质回收率和活性回收率均为４０％左上，由于选用径向色谱柱，色谱参数可直接放大至工业化生产。     

CDld基因稳定转染Panc-1细胞系的建立

实验采用PCR法获得CD1d基因片段,将其插入慢病毒pReceiver-Lv201载体（带GFP荧光）中获得EX-S0249-LV201重组质粒,经转染试剂EndoFectin-Lenti转染到293T细胞中获得慢病毒LP-S0249-LV201,用包装获得的慢病毒感染胰腺癌Panc-1细胞,在不同时间段用倒置荧光显微镜观察绿色荧光,并用反转录酶聚合酶链反应（RT-PCR）和Western blot方法验证获得的表达细胞株.实验结果显示经RT-PCR和Western blotting检测证实慢病毒LP-S0249-LV201 转染至Panc-1细胞株后,该细胞株表达CD1d基因和蛋白,成功建立了稳定表达CD1d基因的Panc-1细胞系,为进一步研究CD1d基因在胰腺癌免疫基因治疗中的作用奠定基础.     

THE EFFECT OF MACROMIXING ON OSCILLATORY BEHAVIOR IN RECOMBINANT FERMENTATION IN A CONTINUOUS STIRRED TANK REACTOR

A general model is presented for recombinant fermentation in a chemostat in which micromixing may be imperfect. Using the production of tryptophan synthetase as an example and the dilution rate, D, as the bifurcation parameter, conditions are first derived for Hopf bifurcation in a perfectly mixed chemostat. The bifurcation surfaces in three-dimensional space are such that oscillatory solutions exist above the surfaces and washout conditions below. A similar analysis is done for imperfect mixing. The bifurcation region is a concave hyperboloid in the [D,f, n]-space, where f and n ; are model parameters. It is argued that poor macromixing reduces the range of D over which bifurcation to periodic solutions is possible.     

重组人肝再生增强因子的纯化及生物学活性分析

目的对在大肠杆菌中表达的重组人肝再生增强因子(rhALR)进行分离纯化及生物学活性分析。方法以大肠杆菌DH5α发酵表达重组人肝再生增强因子,经包涵体洗涤、变性、目的蛋白复性,以离子交换、分子筛等技术纯化,并进行一系列检定。结果目的蛋白以包涵体形式存在,经变性、复性及柱层析纯化,纯度大于98%,SDS-PAGE测定相对分子质量为30000,MOLDI-TOF-MS测定其相对分子质量为30780·522,N-末端15个氨基酸与理论序列一致。Western blot证实rhALR正确表达,等电点5·85~6·85,氨基酸含量与理论值基本符合。动物实验证实rhALR具有明显的促进CCl4毒性肝损伤小鼠肝细胞修复活性。结论经柱层析纯化的rhALR具有促进小鼠CCl4肝损伤肝细胞修复的活性。