基因工程菌的发酵研究

本文对大肠杆菌表达的ｒｈＧＭ－ＣＳＦ工程菌的发酵条件进行了详细的研究,探讨了发酵条件对工程菌表达外源蛋白量的影响,优化了影响发酵的各种条件,形成了一套工程菌发酵表达外源蛋白的工艺,并从工业化角度对工程菌的高密度高表达间的关系进行了探讨。     

基因工程菌发酵生产L-乳酸研究进展

乳酸是重要的工业平台化学品。随着聚乳酸产业的兴起,对高质量L-乳酸的需求量也不断增加。为了进一步降低L-乳酸发酵成本,提高菌株的工业适应性,各种现代生物技术已经应用到L-乳酸发酵菌种的改造上来。文中简要综述了近年来使用乳酸菌、酵母、大肠杆菌及米根霉等基因工程菌株发酵生产L-乳酸的技术进展。     

新型高密度发酵基因工程菌G830

运用PCR方法,从磷酸乙酰转移酶（Pta）－乙酸激酶（Ack)代谢途径缺失菌株E.coliPA1染色体上,扩增出天氨酸激酶－1-高丝氨酸脱氢酶－I（thrA）和高丝氨酸激酶（thrB）基因部分序列,构建了整合型重组质粒pVHb-Kan;应用染色体－质粒同源重组的方法,将透明颤菌血红蛋白（Vitreoscila haemoglobin,VHb)基因整合到大杆菌PA1染色体上的thr操纵子,构建了新型整合工程菌G830。在高密度发酵条件下,G830的细胞呼吸强度、能量代谢、最高菌密度和细胞干重,均明显优于对照菌株PA1和BL21;重组蛋白脯氨酰内肽酶在G830和PA1中获得稳定高表达;重组菌生长状况及发酵指标均与空宿主菌基本一致且表达质粒能维持较好的稳定性。整合型vhb的表达及乙酸代谢途径（Pta-Ack）的缺陷,改善了宿主在贫氧条件下的生长,且促进了重组蛋白的表达。该工程菌具有良好的氧耐受力,且乙酸积累得到大幅度降低,可作为适于高密度发酵的基因工程菌。     

基因工程菌高密度发酵工艺研究进展

阐述了基因工程菌高密度发酵工艺的几个主要影响因素,包括重组菌构建、培养条件、生长抑制因子以及它们的控制技术。通过高密度发酵可以提高细胞生长密度、目的蛋白的表达含量。在高密度发酵过程中,会产生一些有害抑制代谢副产物,但通过分批补料可以降低影响。     

木糖发酵重组菌研究进展

木糖发酵是植物纤维原料生物转化制取乙醇商业化生产的基础和关键 ,但自然界存在的微生物菌株不能满足商业化生产的需要。利用基因工程技术对细菌和酵母进行改造 ,以提高它们在厌氧条件下的木糖发酵能力成为目前研究和开发的重点。通过转基因和基因删除技术 ,主要对Escherichiacoli、Zymomonasmobilis、Pichiastipitis和Saccharomycescerevisiae等典型的乙醇发酵菌株实施基因改造 ,构建出一系列不同类型的木糖发酵重组菌株。与野生型菌株相比 ,重组菌株在厌氧条件下的木糖发酵能力得到了不同程度的改善 ,但是它们仍然未能投入于商业化生产。微生物的木糖代谢工程和木糖发酵重组菌株的构建有待于进一步的深入研究 。     

重组GM-CSF/IL-3融合蛋白表达菌发酵研究

基因工程菌的发酵技术是基因工程药物大规模生产所必备的关键技术,本文对于重组ＧＭ－ＣＤＦ／ＩＬ－３融合蛋白表达菌株Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）（ｐＦｕ）的生长及产物表达规律进行了探索,在此基础上进行高密度发酵研究,真体最终发酵密度达ＯＤ６００值６０以上,目的产物占菌体总蛋白２５％以上。     

基因工程菌的发酵研究

本文对大肠杆菌表达的rhGM - CSF 工程菌的发酵条件进行了详细的研究, 探讨了发酵条件对工程菌表达外源蛋白量的影响, 优化了影响发酵的各种条件, 形成了一套工程菌发酵表达外源蛋白的工艺, 并从工业化角度对工程菌的高密度高表达间的关系进行了探讨。     

重组人白细胞介素-11工程菌的发酵条件研究

为了探讨发酵条件对大肠杆菌表达人白细胞介素-11融合蛋白的影响,利用正交实验设计,对工程菌的生长条件和人白细胞介素-11融合蛋白表达进行优化。在摇瓶中研究了培养基中的葡萄糖、蛋白胨、酵母抽提物的浓度、pH及摇床转速、装液量、接种量等。确定了工程菌生长及表达的培养基和培养条件:葡萄糖10g/L,蛋白胨20g/L,酵母抽提物10g/L,pH7.5,接种量10%,装液量10%,摇床转速220r/min及诱导时间为4~5h。然后在BiofloⅢ-5L发酵罐中以优化的发酵条件进行了3批实验,结果表明:工程菌量达到55g/L(DCW),重组人白细胞介素-11融合蛋白表达量为33%左右,为进行中试研究奠定了理论基础。     

重组截短型人源结缔组织生长因子在大肠杆菌中的表达

用RT－PCR法从人胎盘组织中克隆出人源结缔组织生长因子（h-CTGF)cDNA序列867bp,将此cDNA亚克隆至表达载体pET-9a,重组质粒转化BL21（DE3）pLysS,诱导出N端缺失61个氨基酸残基的截短型rh－CTGF,表达量占总菌体蛋白的7％,主要以不溶性包涵体形式存在,采用离心、洗涤和凝胶过滤分离纯化后,行活性检测表明截短型rt-CTGF无刺激增殖活性,并对用原核表达rt-CTGF作了讨论,为制备抗体、CTGF表达调控和功能研究打下了基础。     

重组人表皮生长因子的稳定性

考察了不同条件下重组人表皮生长因子（rhEGF）的稳定性。结果表明,不同的保存条件（物理状态、温度、pH、浓度等）对rhEGF的稳定性有显影响,使用适当的添加剂（保护剂、抗氧剂、抑菌等）可以提高rhEGF的稳定性。上述结果为rhEGF的制剂学研究提供了依据。     

重组大肠杆菌的高密度发酵和甘油生产条件的初步研究

在摇瓶中进行重组大肠杆菌菌株BL21高密度发酵条件的研究,考察了葡萄糖浓度、盐离子浓度、温度、接种量、发酵时间等对该菌株生产甘油的影响。初步确定底物浓度为2.5%,盐离子浓度0.2%,温度为37℃,接种量为2%,经24h的摇瓶发酵,甘油产量最高达6.8g/L。在30L发酵罐实验中、按初步确定的优化条件发酵26h,甘油产量可达46.67g/L,是LB/葡萄糖培养基中甘油产量的2.06倍。     

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.     

Expression of functional recombinant human factor IX in milk of mice

Human factor IX is synthesized in the liver and secreted in the blood, where it participates in a group of reactions involving coagulation factors and proteins that permit sanguinary coagulation. In this work two lines of transgenic mice were developed to express the FIX gene in the mammalian glands under control of milk β-casein promoter. The founding females secreted the FIX in their milk (3% total soluble protein). The stable integration of transgene was confirmed by southern blot analysis. The presence of the FIX recombinant protein in the milk of transgenic females was confirmed by western blot and the clotting activity was revealed in blood-clotting assays. The coagulation activity in human blood treated with recombinant FIX increased while the time of coagulation decreased. Our results confirm the production of a large amount of recombinant biologically active FIX in the mammary gland of transgenic mice.     

Optimization of a two-stage recombinant fermentation process: the dilution rate effect

The effects of dilution rates on the performance of a two-stage fermentation system for a recombinant Escherichia coli culture were studied. Dilution rate determines the apparent or averaged specific growth rate of a heterogeneous population of cells in the recombinant culture. The specific growht rate affects the genetic parameters involved in product formation in the second stage, such as plasmid stability, plasmid content, and specific gene expression rate. Kinetic models and correlations were developed for these parameters based on experimental data. Simulations of plasmid stability in the first stage showed that for longer fermentation periods, plasmid stability is better at higher dilution rates. However, the plasmid content is lower at these dilution rates. The optimal apparent specific growth rate for maximum productivity in the second stage was determined using two methods: (1) direct search for a constant specific growth rate, and (2) dynamic optimization using the maximum principle for a time-dependent specific growth rate profile. The results of the calculations showed that the optimum constant apparent specific growth rate for maximum over-all productivity is 0.40 h(-1). This coincides with the optimal specific growht rate for maximum plasmid content in the expressed stage. A 3.5% increase in overall productivity can be obtained by using a linear time dependent apparent specific growth rate control, mu(2)(t) = 0.0007t, in the course of the fermentation time.     

海藻糖产生菌的筛选及其发酵条件研究

通过液体发酵筛选,从土壤中分离的241株菌株及保藏的95株菌株中得到15株海藻糖生产菌,对这些菌株进一步筛选得到产海藻糖最多的一株菌Bacillus sp。其发酵最适合条件为：以麦芽糖为碳源,发酵初始pH值7.0,接种量为6%,30℃条件下培养48h,另外该菌株在分别以葡萄糖和蔗糖为碳源的培养基中都能产生海藻糖。     

Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

In this study the w/o/w extraction–evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The micro-spheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical micro-spheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the mi-crosphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.