重组人尿型纤溶酶原激活剂的中试生产与性质研究

表达uPA的CHOdhfr工程细胞CL11G在30升生物反应器中高密度大规模培养65天后,获得1200余升细胞培养上清;经阳离子交换层析、凝胶过滤、苯甲眯亲和层析和阴离子交换层析四步纯化工艺,获得uPA冻干纯品40克。对产品进行性质研究和质量检定,各项指标符合理论数据和《中国生物制品规程》。     

面包树果实凝集素的提取纯化及理化性质分析

建立面包树果实凝集素(frutalin,FTL)的提取纯化方案,并对FTL纯品进行部分理化性质分析。本试验以新鲜的面包树果实籽为材料,经Tris-HCl缓冲液浸提、过滤、硫酸铵沉淀和阳离子交换层析后超滤浓缩得FTL纯品,最后用纯品开展了糖抑制,热稳定性,pH稳定性和金属离子耐受性等试验。提取优化得到的最佳条件为:料液比1∶2(g:m L),浸提时间2 h,浸提温度50℃,硫酸铵饱和度70%,沉淀时间4 h。经阳离子交换层析后FTL回收率为22%。研究结果表明,FTL是一种热稳定性凝集素,在温度不超过60℃时,其凝血活性不受影响,并且不依赖于金属离子来发挥其凝血活性。本研究建立了一个简单高效的FTL分离纯化方案,并对FTL纯品做了部分理化性质研究,为FTL可能的生物医学应用提供研究基础。     

白眉蝮蛇蛇毒神经生长因子的研究Ⅰ.神经生长因子(NGF)的纯化及其生物学活性鉴定

研究白眉蝮蛇蛇毒神经生长因子纯化方法并行生物学活性鉴定。方法从白眉蝮蛇乌苏里亚种蛇毒中,经硫酸胺分段盐析,提取含有神经生长因子的粗制品,再经二乙氨基乙基纤维素离子交换层析和羧甲基纤维素离子交换层析,分离出蛋白质纯品,经十二烷基磺酸钠——聚丙烯酰胺凝胶电泳进行鉴定,结果纯化的纯品为单一区带,分子量为４３ｋｕ。生物学活性鉴定证明分离出的该蛋白质纯品可促进神经细胞突起密集生长。     

一种白细胞介素2基因变构体的高效表达与纯化工艺

利用定点诱变技术构建表达质粒pET15b-MhIL-2并将其在大肠杆菌中进行表达发酵的优化研究,高效表达出可溶性的MhIL-2重组蛋白。蛋白经过亲和层析、Thrombin酶切、离子交换层析和凝胶过滤层析纯化,MhIL-2纯度达95%,且MhIL-2比hIL-2具有更强的促进T细胞增殖生物活性。     

人甲状旁腺激素在大肠杆菌中的表达及鉴定

化学合成人甲状旁腺激素（hPTH）全长基因,克隆到大肠杆菌表达载体pBV220和pET22b中,获得了高表达。经破菌、阳离子交换层析、反相层析纯化获得了纯度大于95%的纯品。N端测序、质谱分析结果表明重组hPTH 结果完整,N端无Met或fMet。生物活性试验证明重组hPTH具有激活腺苷酸环化酶、增加骨质量和骨密度等作用。     

纳豆激酶的分离纯化及生化研究

用固体发酵工艺制得的纳豆中含有大量溶栓活性成分———纳豆激酶。经过生理盐水浸提 ,(NH4 ) 2 SO4 分级沉淀 ,Sephadex柱层析等步骤 ,可以得一层析纯的纳豆激酶。冷冻干燥品经SDS—PAGE电泳显示有二组分存在     

L-精氨酸发酵条件的研究

本文报道了产L一精氨酸突变株(Corynebacterium crenatum)971．1(SG,His-)摇瓶发酵条件的研究结果。试验结果表明,培养基中组氨酸与生物素的适宜用量有助于产物的生成,组氨酸或豆饼水解物用量分别为50~g／’ml和o．{％、生物素或玉米浆用量分别为7 5—125 pg／L和2．0％时,可得到较高产酸率。为获得较高的精氨酸积累量,培养基中初糖浓度为12％,硫l酸铵用量应高达6．5％为适宜,如减少摇瓶装液量以改善通风状况可明显提高产酸率。在适宜的发酵条件下,971．1菌株经发酵培养96小时,产酸最高可达到3{mg／mlo发酵液经732型离子交换树脂提取后,获得纯品结晶。该结晶经生物鉴定、比旋度测定以及c、H、N元素含量分析,其结果均与文献值相符;红外光谱分析与标准样品一致;纸层析图谱表明,50pg样品层析显色后为单斑点,其＆值与标准品相同;从而证明所得纯品结晶是L一精氨酸。     

一种新型肿瘤细胞抑制蛋白的分离纯化

介绍了从健康牛骨髓中分离到一种具有抑制肿瘤活性的糖蛋白,并对该糖蛋白进行了纯化,获得了电泳纯和层析纯的纯品．测得该糖蛋白N末端为两氨酸残基．用SDS-PAGE法测得该溏蛋白的表现分子量为65kD．氨基酸组成分析显示其为一种富含丝氨酸的蛋白质．生物活性测定表明,该糖蛋白纯品对小白鼠白血病P388细胞和人慢粒白血病HL-60细胞株的增殖均有抑制作用．有关该糖蛋白的糖含量的定量测定及蛋白质一级结构分析正在进一步的研究中．     

重组人干扰素α2b的制备研究

为了获得高活性高纯度的rh IFNα2b,对重组人干扰素α2b进行克隆、表达,并深入研究了其纯化工艺。采用重叠延伸PCR法合成了编码IFNα2b的基因,用DNA重组技术构建了原核表达载体p BV220-IFNα2b,获得了稳定的工程菌种。发酵产物通过破菌、洗涤获得包涵体,再经过变性、复性、离子交换层析和凝胶过滤层析的纯化,得到rh IFNα2b纯品,其比活可达1×10~8IU/mg。实验结果为进一步开展临床前研究和长效制剂奠定了基础。     

蜘蛛杀虫肽与Bt-toxin C肽融合蛋白(BGT)基因的原核表达与蛋白质纯化

从质粒pCAMBIA2301-BGT中获得蜘蛛杀虫肽与Bt-toxin C肽融合蛋白(BGT)基因编码区全长,并构建了原核表达载体pET28a-BGT。将此质粒转入大肠杆菌BL21(DE3)中,获得有效表达,新蛋白的分子量约为51 kDa,主要以包涵体形式存在。在变性条件下以不同的咪唑和pH值洗脱方式进行比较,确定以咪唑为金属鳌合亲和柱层析的洗脱条件,获得了纯BGT蛋白。     

Optimization of fermentation conditions for an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain engineered using the response surface method to produce a novel therapeutic DNA vaccine for rheumatoid arthritis

Background

Fermentation condition optimization and nutrients screening are of equal importance for efficient production of plasmid DNA vaccines. This directly affects the downstream purification and final quality and yield of plasmid DNA vaccines. The present study aimed to optimize the fermentation conditions for high-throughput production of therapeutic DNA vaccine pcDNA-CCOL2A1 by engineered Escherichia coli DH5α, using the response surface method (RSM).

Results

We hypothesized that optimized fermentation conditions significantly increase the yield of pcDNA-CCOL2A1 therapeutic DNA vaccine, a novel DNA vaccine for treating rheumatoid arthritis (RA). Single-factor analysis was performed to evaluate the optimal basal culture medium from LB, 2?×?YT, TB, M9 (Glycerol) and M9 (Glucose), respectively. Thereafter, the Plackett-Burman design (PBD) was used to ascertain the three most significant factors affecting the vaccine yields, followed by the paths of steepest ascent to move to the nearest region of maximum response. Initial screening through the PBD revealed that the most key factors were peptone, mannitol, and inoculum concentration. Subsequent use of RSM was further optimized for the production of therapeutic DNA vaccine pcDNA-CCOL2A1 through Box-Behnken design (BBD). The final optimized fermentation conditions were as follows: peptone, 25.86 g/L; mannitol, 8.08 g/L; inoculum concentration, OD?=?0.36. Using this statistical experimental design, the yield of therapeutic DNA vaccine pcDNA-CCOL2A1 markedly increased from 223.37 mg/L to339.32 mg/L under optimal conditions, and a 51.9% increase was observed compared with the original medium.

Conclusions

The present results provide a basis for further production of high-quality and high-yield therapeutic DNA vaccine pcDNA-CCOL2A1 in pilot-scale and even industrial-scale.

     

Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli

Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380?mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86?mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.     

Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations

Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption-desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Plasmid DNA vaccine vector design: Impact on efficacy,safety and upstream production

Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance's are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for follow-on products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.     

一株维氏气单胞菌发酵培养基优化及其制备疫苗免疫效力比较

采用单因素试验、响应面试验法对维氏气单胞菌（Aeromonas veronii）发酵培养基的氮源、碳源、无机盐和磷酸盐成分及用量进行优化组合,确定优化培养基组成：胰蛋白胨10.8 g/L,葡萄糖5.0 g/L,牛肉膏3.0 g/L,磷酸二氢钾2.0 g/L,硫酸镁0.4 g/L,NaCl 5.0 g/L。并与基础培养基的发酵活菌数、制备的灭活疫苗免疫效力进行比较,经过验证试验绘制维氏气单胞菌在优化培养基条件下的7 L发酵罐生长曲线。在优化发酵培养基条件下,维氏气单胞菌活菌数为5.94×10⁹ cfu/mL,比基础培养基增幅43.13%;制备的灭活疫苗相对保护率为77.78%,比基础培养基提高了14.81%。7 L发酵罐发酵培养10 h,活菌数达到最大8.85×10⁹ cfu/mL。通过对发酵培养基的优化,可以获得低成本、优质高效的维氏气单胞菌发酵菌液,为今后维氏气单胞菌灭活疫苗规模化发酵培养提供参考。     

Gluconobacter oxydans木糖醇脱氢酶基因的克隆表达及木糖醇的转化分析

从氧化葡萄糖酸杆菌(Gluconobacter oxydans)的基因组DNA上扩增出木糖醇脱氢酶基因xdh,构建了诱导型表达载体pSE-xdh,导入E.coli JM109后获得了高效表达木糖醇脱氢酶基因的重组菌JM109/pSE-xdh。通过HisTrap HP亲和层析和SephacrylS 300分子筛两步纯化从细胞中得到纯酶,并对酶学性质进行研究。XDH最适还原反应的pH值为5.0,最适还原反应的温度为35℃;最适氧化反应的pH值为11.0,最适氧化反应的温度为30℃。重组菌中的XDH依赖NADH,对NADH的米氏常数Km=57.8 mmol/L,最大反应速率Vmax=1209.1 mmol/(ml·min)。重组菌的XDH酶活力为13.9 U/mg。利用重组菌和原始菌混合静止细胞转化D 木酮糖,16 h 28.0 g/L D木酮糖生成16.7 g/L木糖醇,而原始菌单独转化只生成8.3 g/L木糖醇。     

The role of amino acids in the amplification and quality of DNA vectors for industrial applications

In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed-batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications.     

Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.