乳糖诱导方式对降血糖药物表达的影响

对重组E.coli BL21(DE3)表达降血糖药物胰升血糖素样肽-1类似物的乳糖诱导方式进行了研究.结果表明诱导剂乳糖分批添加在生物量与蛋白表达量上均优于一次性添加方式,因为乳糖可被细菌作为碳源利用,分批添加乳糖诱导的目的蛋白表达量高达48.0%.研究结果证明乳糖分批流加可用于高密度发酵表达重组GLP-1类似物,并对其它重组基因工程药物的生产具有指导意义.     

重组大肠杆菌高密度发酵表达胰高血糖素样肽-1衍生物(GP62)的培养基优化

对重组E.coli BL21 (DE3)高密度表达胰高血糖素样肽-1衍生物(GP62)的发酵培养基进行了优化.通过单因素实验与正交实验相结合的方法,依次对基础培养基,发酵培养基中碳源、氮源、无机盐等成份进行优化.在2YT培养基基础上,获得优化的发酵培养基2YT-GP62.在5L全自动发酵罐中利用终浓度0.5 mmol/...     

The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell

Control of mammalian gene promoters by the bacterial LacI repressor provides reversible regulation and dose-response levels of derepressed expression by the lactose analog isopropyl thiogalactose (IPTG). Here, we show that insertion of LacI-binding sites in the ubiquitous β-actin promoter confers a strong and dose-dependent IPTG-regulatable expression of transiently transfected reporter genes in mouse embryonic stem (ES) cells expressing LacI. We established ES cell lines stably expressing reporter genes under inducible control and found a five- to tenfold IPTG induction of transgene expression. The kinetics of induction is rapid and stable, and can be rapidly reversed after IPTG removal. Importantly, this regulatable expression was maintained throughout the differentiation process of ES cells, and observed in individual differentiated cardiomyocyte-like cells and neuronal-like cells. This reversible system is the first to function from undifferentiated to individual welldifferentiated ES cells, providing a very useful tool to understand molecular mechanisms underlying ES cell self-renewal, commitment and differentiation.Received 17 March 2005; received after revision 19 April 2005; accepted 25 April 2005     

大肠杆菌工程菌发酵工艺中诱导条件的研究

通过比较在分批发酵中分别加入诱导剂IPTG0．3mM和0．5mM与诱导时间为3h和4h的不同组合,观察其对重组蛋白表达的影响,从而筛选出目的蛋白高效表达的最佳条件．实验表明,工程菌在对数生长的中后期（OD600为4）时添加终浓度为0．5mM的WIG诱导对蛋白的表达最为有利．     

乳糖诱导大肠杆菌中重组谷氨酰胺合成酶的表达

以重组枯草芽孢杆菌谷氨酰胺合成酶（L-谷氨酸：氨连接酶,Glutamine Synthetase,GSEC6.3.1.2）蛋白表达宿主菌株BL21（DE3）（pET3C／谷氨酰胺合成酶）作为研究对象,借助SDS-PAGE分析方法,对于用乳糖诱导由T7lac启动子控制的重组目的产物蛋白的表达规律进行了优化研究,分别比较了最佳诱导起始生长量、所需乳糖的浓度、诱导持续时间、补加乳糖和氨苄青霉素及不同培养基等参数对重组产物表达的影响．实验结果表明,对于T7lac启动子控制的重组目的产物,乳糖作为诱导剂也可以起到很好的诱导表达效果,诱导产物的表达量、可溶性及酶活与IPTG诱导产物基本接近．本研究结果为乳糖作为诱导剂应用于重组大肠杆菌谷氨酰胺合成酶工业化生产提供了一定的参考依据．     

乳糖诱导pET载体表达重组蛋白的研究

以重组人B淋巴细胞刺激因子（hBLyS）蛋白表达宿主菌株BL21（DE3）（pET30(a)/hBLyS）作为研究对象，借助SDS－PAGE分析方法，对于用乳糖诱导由T7lac启动子控制的重组目的产物的表达规律进行了优化研究。分析比较了诱导的起始阶段、所需的乳糖浓度和诱导持续时间等参数对重组产物表达的影响。实验结果表明，对T7lac启动子控制的重组目的产物，乳糖可以作为诱导剂；在对诱导条件进行优化控制的前提下，乳糖诱导目的产物的绝对表达量虽然不及IPTG，但相对成本却比IPTG低得多。研究结果为乳糖作为诱导剂应用于重组基因工程药物工业化生产提供了一定的参考。     

Cloning and molecular characterization of a gene encoding MAP kinase from maize and its expression in E. coli

A new MAPK gene, ZmSIMK1 （Zea mays L. salt-induced mitogen-activated protein kinase 1）, is isolatod from a maize eDNA library. The full-length ZmSIMK1 gene contains 1636 bp and an open reading frame of 1122 nucleotides capable of eneoding 373 amino acid polypeptides with a predicted molecular mass of 42.3 kda and pI of 6.01. The putative ZmSIMK1 protein contains all 11 conserved subdomains that are characteristics of serine/threonine protein kinases and the TEY motif, which is the putative phosphorylation site. Northern blot analysis shows that ZmSIMK1 is ubiquitously expressed in roots, stems, and leaves of maize seedlings and its mRNA accumulation is observed in maize seedlings treated with 30 mmol/L PEG-6000 and 137 mmol/L NaCl, but the expression of ZmSIMK1 is not significantly affected by 4℃ treatment. The expression vector pET-ZmSIMK1 is constructed by inserting the coding region of ZmSIMK1 eDNA into pET-42a（＋）, and transformed into E. coli strain BL21（DE3）. A 77kda fusion protein is induced by the further culture at 37℃ after addition of 1 mmol/L IPTG.     

重组人B淋巴细胞刺激因子原核表达条件的优化

以重组人B淋巴细胞刺激因子(rhBLyS)蛋白表达宿主菌株M15(pQE30a／rhBLyS)作为研究对象，借助SDS-PAGE分析方法，对于用IPTG诱导的重组目的产物的表达进行了优化研究。分析比较了pH值、裂解液种类、诱导时问、IPTG浓度、温度等参数对重组产物表达的影响．实验结果表明，降低温度和IPTG浓度有利于增加靶蛋白的可溶性，27℃和0．075mol／L IPTG为比较适合的表达条件．