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为了去除乙酸对rIL-2工程菌生长及基因表达的抑制作用,用医用人工肾作为透析器建立了一套透析培养装置,通过该装置对rIL-2工程菌进行了透析培养的研究,结果表明透析液、pH、透析培养的持续时间段对工程菌的生长及rIL-2的表达有较大影响。通过选择高浓度的无机盐透析液及诱导后期的透析培养方法使菌密度在50 A_(600nm)时rIL-2表达水平仍大于40%。 相似文献
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乙酸积累对基因工程菌培养的影响及与培养基pH的关系 总被引:7,自引:0,他引:7
在rIL-2工程菌K_(802)(pLY—4)高密度培养中,发现培养液中有大量代谢副产物乙酸积累,乙酸的存在对工程菌的生长和产物的表达均有明显的抑制作用,这种抑制作用是制约工程菌高密度培养的重要因素。为了减小这种抑制作用,初步研究了培养基pH与乙酸抑制作用的关系,发现适当提高培养基pH值,能减小乙酸的抑制作用;高密度培养时,提高培养基的pH后,虽然仍有大量乙酸积累,但产物的表达水平和菌密度都有提高。 相似文献
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在确定了培养基及pH值的基础上,进一步观察了升温诱导过程中有机酸的产生及其对工程菌E.coli DH5α(pHV-hIL-6)生长和rIL-6表达的影响。当有机酸浓度低于70mmol/L以下时,菌密度达到干重2~3.5g/L之间收菌,rIL-6的表达水平为25%~32%;当有机酸浓度达到70mmol/L以上时,工程菌的生长不受影响,而rIL-6的表达明显受抑制。产生的有机酸以乙酸为主。收集菌体后,经过破菌,分离提纯的包涵体,其rIL-6的纯度可达到70%。用GuHCl缓冲液溶解包涵体,样品稀释后经过Q Sepharose F F柱纯化,可得到纯度达95%以上的rIL-6。采用依赖IL-6的小鼠杂交瘤细胞系7TD1及MTT比色法测定生物活性,rIL-6的比活性为2×10~8U/mg。 相似文献
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以表达普鲁兰酶的重组大肠杆菌作为出发菌株,在摇瓶培养的基础上,建立了大肠杆菌工程菌产普鲁兰酶的高密度发酵工艺。通过测定细胞密度、细胞干重、分离菌体可溶性成分与不溶性成分及SDS-PAGE电泳,分析重组大肠杆菌的生长和普鲁兰酶的表达情况。摇瓶试验使用合成培养基和LB培养基,重组大肠杆菌在合成培养基生长较慢,诱导5 h的普鲁兰酶表达量高于LB培养基,包涵体比例低于LB培养基。重组大肠杆菌的高密度发酵使用合成培养基,补料阶段采用指数流加的工艺,在设定细胞的比生长速率为0.12的前提下,限制补料中碳源的供应,以阻止乙酸的产生。当细胞密度OD600达到70.0开始诱导,最终细胞密度为每升53.3 g细胞干重,细胞内可溶性普鲁兰酶为每升1.35 g。 相似文献
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生长速度对β干扰素工程菌稳定性和产物表达的影响研究 总被引:1,自引:1,他引:0
为了研究发酵培养阶段生长速度对重组人干扰素β(hIFN-β)工程菌稳定性、菌体密度和干扰素表达的影响,采用日本LE.Marubishi MSJ-50L发酵罐,在发酵过程中于诱导后通过控制不同的补料速度,检测低和高生长速度及发酵结束后菌体密度、稳定性和重组人干扰素β的表达量。诱导后维持低生长速度,发酵结束后平均密度A600值为7.52,表达量达20.7%,质粒稳定率好,达到91%,粗制干扰素收获2.37克;维持高生长速度,发酵结束后平均密度A600值达20.57,而表达量降为4.13%,质粒稳定率降为27%,粗制干扰素收获仅为0.59克。诱导后生长速度对工程菌稳定性和产物表达有明显影响。 相似文献
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根据重组人碱性成纤维细胞生长因子基因工程菌的生长特点,对其高密度发酵工艺条件进行研究和改进,采用“平衡DO-State”控制策略进行分批补料培养中的葡萄糖流加,有效地控制了培养过程中代谢副产物-乙酸的产生及其对工程菌生长的抑制作用,使发酵终了时乙酸浓度由15.6g/L下降为2.6g/L,而菌体密度则由15.2gDCW/L提高到30.2gDCW/L。 相似文献
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抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究 总被引:6,自引:0,他引:6
denoregulin(ADR)是来源于南美树蛙Phyllomedusa bicolor皮肤的含有33个氨基酸的抗菌肽,在非极性环境中形成α_螺旋型结构,具有抗菌活性强、抗菌谱广的特点。将ADR基因克隆于pET32a载体上,转化大肠杆菌BL21(DE3),对这一工程菌株的培养条件进行了优化。通过正交试验,考察诱导时机、诱导剂量和诱导时间三个因素的不同水平对蛋白表达的影响,结果发现诱导时机的影响尤为显著,考察了9种不同培养基对表达量的影响,发现培养基中加入葡萄糖对目标蛋白的稳定表达起了重要的作用,确定最佳培养条件为:培养基为2×YT+0.5%葡萄糖,诱导时机为OD600=0.9左右,诱导剂IPTG加入的终浓度为0.1mmol/L,诱导时间为4h。采用前期恒pH、后期指数流加的策略进行工程菌BL21(DE3)/pET32a-adr的高密度培养,在整个流加过程中,通过控制葡萄糖的加入量,将菌株的比生长速率控制在015h-1,乙酸浓度也被控制在较低的水平(<2g/L),但是质粒丢失严重,在发酵结束时,约有40%的大肠杆菌中不带质粒,这导致了目标蛋白的表达量下降严重,但是表达的目标蛋白90%以上为可溶性形式。表达的融合蛋白无抑菌活性,而裂解后得到的ADR单体具有明显的抑菌活性。 相似文献
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XUZIQIN JINGFENJIA 《Cell research》1995,5(2):187-195
An efficient protocol for plant regeneration from protoplasts of hydroxyproline(HYP)resistant cell line of Onobrychis viciaefolia was established.In SH medium supplemented with 1mg/L2,4-dichlorophenoxy-acetic acid(2,4-D),0.5mg/L kinetin(KT)and 0.2mg/L naphthalene acetic acid(NAA),the division frequency of protoplastderived cells reached up to over 60%,and microcalli were obtained in 5-6wk.Upon transferring them on agar solidified MS medium plus 2mg/L indole-3-acetic acid (IAA),shoots were induced.After cultivating them on MS medium with or without IAA,roots were regenerated.Chromosome number of all protoplast-regenerated plants examined were normal(2n=28).The protoplast-derived calli and plants grew vigorously on the medium containing 10 mmol/L HYP. 相似文献
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Regulation of Fc receptor for IgE (CD23) and class II MHC antigen expression on Burkitt's lymphoma cell lines by human IL-4 and IFN-gamma 总被引:10,自引:0,他引:10
F Rousset R W Malefijt B Slierendregt J P Aubry J Y Bonnefoy T Defrance J Banchereau J E de Vries 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(8):2625-2632
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INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode… 相似文献
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High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor 总被引:3,自引:0,他引:3
A recombinant Escherichia coli HB101(pPAKS2) producing penicillin acylase was cultured in a membrane cell recycle fermentor. The strain was very stable throughout the whole experiment. The main inhibitory by-product was acetic acid, and cell growth ceased when its concentration was above 14 g/L Cell density could be increased up to 145 g/L dry weight without experiencing by-product inhibition by regulating glucose concentration in the fermentor and by using total membrane recycle. Acetic acid formation was negligible not only when cells were cultured in medium containing no glucose but also when glucose was limited. Dissolved oxygen control as well as glucose limitation was an indispensable condition for minimizing acetic acid formation when the medium contained glucose. Low concentrations of accumulated acetic acid were reused when glucose was limited. Use of highly concentrated medium reduced the membrane surface area required for cell recycle greatly. The recycle fermentor could be operated in various operational modes including partial bleed and repeated recycle culture to give high productivity. Productivity of a repeated recycle system was over 10 times higher than that of a simple batch system. 相似文献
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将骆驼刺离体再生苗的茎切段经发根农杆菌A4菌株感染后,在含500mg/L头孢霉素的MS无激素培养基上培养,产生了转化的发根和愈伤组织.转化根在附加2mg/L2,4-D和0.5-1mg/L6BA的MS培养基上培养后,亦可诱导出愈伤组织.在含3mg/L6BA和0.5mg/LNAA的培养基上诱导出了苗的分化.冠瘿碱分析表明,在95%以上的发根和75%的转化愈伤组织及再生植株中都显示了T-DNA的整合和表达.染色体检查发现,约81%的发根细胞具有正常染色体数(2n=18),其余则存在染色体数目的变化,在继代培养一年之后,转化体仍维持旺盛的再生能力. 相似文献
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Bernard Pélissier Ouafa Bouchefra Régis Pépin Georges Freyssinet 《Plant cell reports》1990,9(1):47-50
We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.Abbreviations NAA
1-naphthalene acetic acid
- IAA
indole-3-acetic acid
- BAP
6-benzylamino-purine
- MS
Murashige and Skoog medium
- B5
Gamborg medium 相似文献