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目的：对毕赤酵母重组菌GS115-Ch—Glu菌种进行发酵脱毒条件的优化,为大规模发酵重组毕赤酵母获得高表达目的蛋白奠定基础。方法：以毕赤酵母重组菌为实验菌株,分别以葡萄糖、乳糖、玉米淀粉、蛋白胨、甘油、玉米淀粉＋甘油为碳源,研究不同碳源对菌株细胞生长的影响。结果：毕赤酵母重组菌的最适碳源为玉米淀粉,最适浓度为2．5％。 相似文献
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巴斯德毕赤酵母表达系统是应用较为成功的外源蛋白表达系统之一,它具有真核生物表达系统的特点,能够对目的蛋白进行类似高等真核生物的信号肽剪切、二硫键形成、糖基化等过程的翻译后蛋白加工,至今已有多种外源蛋白在该表达系统中成功表达.本文综述了毕赤酵母表达系统的一些特点和优势、毕赤酵母表达载体组成和外源蛋白在毕赤酵母中表达的过程. 相似文献
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将人细胞周期蛋白D1基因克隆入原核表达载体pET-20b中获得重组质粒pET-20b-eyeD,经酶切鉴定正确后转化大肠杆菌BL21PlaysS后获得表达菌株．该菌株经IPTG诱导后表达的目的蛋白有部分分泌到培养基上清中,将培养基上清中的蛋白沉淀后用Ni^2＋螯合柱进行纯化,最后可得到纯度达到95％以上的目的蛋白．蛋白电泳显示纯化蛋白的分子量约为33KD,Westemblot分析表明,在电泳胶的相应分子量处出现特异性条带,说明已经成功表达和纯化了重组人细胞周期蛋白D1． 相似文献
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人酸性成纤维细胞生长因子cDNA的表达 总被引：2，自引：0，他引：2
目的：研究人酸性成纤维细胞生长因子（haFGF）cDNA在不同表达系统中的表达，旨在得到高效表达haFGF的表达系统。方法：采用PCR方法扩增人酸性成纤维细胞生长因子(haFGF）cDNA，将该cDNA片段分别插入表达载体pKK223-3及pET3C的表达框架中，筛选得到了重组质粒pKK223-3-haFGF及pET3C-haFGF，分别转化大肠杆菌JM109及BL21（DE3），构建了表达菌株JM109/pKK223-3-haFGF及BL21(DE3)/pET3C-haFGF,用IPTG诱导表达。结果；SDS-PAGE的结果表明，表达菌株BL21(DE3)/pET3C-haFGF能高效表达人酸性成纤维细胞生长因子，而JM109/pKK223-3-haFGF诱导后目标蛋白带不明显。结论：BL21（DE3）/pET3C表达系统更适于表达人酸性成纤维细胞生长因子。 相似文献
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真核生物表达系统研究进展 总被引：4，自引：0，他引：4
郑光宇 《喀什师范学院学报》2004,25(6):33-36
从毕赤酵母表达宿主菌，表达载体，外源蛋白在毕赤酵母中的表达，毕赤酵母表达系统的优化和丝状真菌的转化，CBH表达系统及rDNA整合序列等方面综述了近年来关于毕赤酵母和丝状真菌表达系统的研究进展． 相似文献
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1IntroductionNeural stemcells(NSCs)are a subtype of progenitorcells in the nervous systemthat can differentiate intoneurons and glia[1-3].Due to their feature of self-re-newal,NSCs have expectations for treatment of ner-vous system diseases such as Parkin… 相似文献
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采用PCR方法克隆获得拟穴青蟹抗菌肽scygonadin基因成熟肽片段,将其连接到Pichia pastoris酵母表达载体pPIC9K中,构建了分泌表达载体pPICgk-Scy．电击转化毕赤酵母GS115,经表型筛选和PCR鉴定证实了目的基因已稳定整合人酵母基因组中．以甲醇诱导表达阳性克隆,上清中表达产物经过Tricine-SDS-PAGE鉴定与预期的目的蛋白大小（约10ku）相符．并利用琼脂孔穴扩散法证明了表达产物能抑制革兰氏阳性菌和革兰氏阴性菌的生长． 相似文献
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研究用亲和融合谷胱甘肽 S 转移酶 (GST)的方法纯化重组人白细胞介素 6(IL 6)的发酵和纯化工艺 ,使用含有质粒pHZl818的E .coliJMl0 9在 2XYT培养基中进行发酵表达 ,IL 6表达为与谷胱甘肽 S 转移酶 (GST)融合的融合蛋白GST IL 6.融合蛋白存在可溶的活性蛋白和不可溶的包含体两种形式 ,此包含体无活性且无法复性 ,无法用亲和层析回收 ,通过实验优化摇床发酵的诱导温度和转速 ,以增加可溶融合蛋白的表达 .菌体超声破碎液后 ,上清液用作亲和柱层析 ,可将融合蛋白提纯至 80 00 ,每升发酵液可得 10mg融合蛋白 ,用凝血酶裂解处理 6h ,亲和标志物GST被特异性切除 ,裂解得到的IL 6用离子交换柱层析可纯化至 95 00 ,MTT法测得纯化的IL 6生物学活性为 1.0 2× 10 8IU/mg . 相似文献
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Zhong-hui ZHANG Li-juan DU Di XIANG Shun-ying ZHU Ming-yuan WU Hui-li LU Yan YU Wei HAN 《Journal of Zhejiang University. Science. B》2009,10(2)
Midkine is a heparin-binding growth factor,which plays important roles in the regulation of cell growth and differentiation.The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor.In the present work,rhMK was expressed in Escherichia coli (E.coli) BL21 (DE3).The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG).After sonication,midkine was recovered in an insoluble form,and was dissolved in guaoidine hydrochloride buffer.Renaturation of the denatured protein was carried out in the defined protein refolding buffer,and the refolded protein was purified using S-Sepharose ion-exchange chromatography.The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC).The purified rhMK enhanced the proliferation of NIH3T3 cells. 相似文献
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绿僵菌是一类广泛应用于生物防治的昆虫病原真菌.研究表明小RNA能够调控基因的表达,其中Argonaute基因在整个小RNA通路中发挥重要的作用.本研究通过RT-PCR的方法从绿僵菌中获得了Argonaute基因的部分功能片段,构建了其重组原核表达载体,将重组载体转化至大肠杆菌进行诱导表达;采用镍柱亲和纯化重组的目的蛋白并通过Western blot技术鉴定.结果发现：通过RT-PCR的方法获得长度约为950bp的基因片段;重组原核表达载体经诱导表达后,SDS-PAGE检测发现分子量约为34kDa的目的蛋白条带;诱导5h后蛋白的表达量最高,采用镍柱亲和层析纯化重组蛋白,经Western blot技术检测,重组蛋白可与His-tag抗体发生特异性反应.该纯化重组蛋白的获得为将来绿僵菌Argonaute蛋白抗体的制备,并进一步通过该抗体获得绿僵菌体内的小RNA及其靶基因提供了基础. 相似文献
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ＩＮＴＲＯＤＵＣＴＩＯＮＦｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ (ＦＧＦ)ｉｓａｎａｎｇｉｏ ｇｅｎｉｃａｎｄｍｉｔｏｇｅｎｉｃｐｏｌｙｐｅｐｔｉｄｅｔｈａｔｃａｎｐｒｏｍｏｔｅｃｅｌｌｐｒｏｌｉｆｅｒａｔｉｏｎａｎｄｄｉｆｆｅｒｅｎｔｉａｔｉｏｎｉｎａｗｉｄｅｖａ ｒｉｅｔｙｏｆｃｅｌｌｔｙｐｅｓ (Ｇｏｌｄｆａｒｂ ,1 990 ;Ｂａｓｉｌｉｃｏｅｔａｌ.,1 992 ) .ＴｈｅＦＧＦｆａｍｉｌｙｈａｓｍａｎｙｍｅｍｂｅｒｓｗｉｔｈｓｉｍｉｌａｒａｍｉｎｏａｃｉｄｓｅｑｕｅｎｃｅａｎｄｂｉｏｌｏｇｉｃａｌａ… 相似文献
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Human acidic and basic fibroblast growth factors (aFGF and bFGF) are classic and well characterized members of the heparin-binding
growth factor family. Heparin is generally thought to play an extremely important role in regulating aFGF and bFGF bioactivities
through its strong binding with them. In order to unravel the mechanism of the interactions between heparin and FGFs, and
evaluate the importance of heparin sulfate groups' binding with FGFs, surface plasmon resonance analyses were performed using
IAsys Cuvettes System. Heparin and its regioselectively desulfated derivatives were immobilized on the cuvettes. aFGF and
bFGF solutions with different concentrations were pipetted into the cuvettes and the progress of the interaction was monitored
in real-time by Windows-based software, yielding kinetic and equilibrium constants for these interactions. In addition, in
order to reduce the delicate difference among the cuvettes, inhibition analyses of mixtures of FGFs and immobilized native
heparin by modified heparins were also done. The data from these two methods were similar, indicating that all sulfate groups
at 2-O, 6-O and N- in heparin were required for the binding to aFGF; and that their contribution to the binding was in the
order 2-O, N- and 6-O-sulfate group. In contrast, definite contribution of the 6-O-sulfate group to the binding with bFGF
was most apparent, while the other two sulfate groups appeared to be necessary in the order 2-O and N-sulfate group. These
methods established here can be used for analysing the effect of sulfate groups in heparin on the binding with other human
FGF members or other heparin-binding proteins.
The project supported by the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry
of China and Zhejiang Provincial Natural Science Foundation of China (301306) 相似文献
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Zhong-hui Zhang Li-juan Du Di Xiang Shun-ying Zhu Ming-yuan Wu Hui-li Lu Yan Yu Wei Han 《Journal of Zhejiang University. Science. B》2009,10(2):79-86
Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation.
The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important
growth factor. In the present work, rhMK was expressed in Escherichia coli (E. coli) BL21 (DE3). The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG). After sonication,
midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured
protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange
chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid
gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC). The purified rhMK enhanced
the proliferation of NIH3T3 cells.
Project supported in part by the Hi-Tech Research and Development Program (863) of China (No. 2007AA02Z149) and the Science
and Technology Commission of Shanghai Municipality (No. 075407071), China 相似文献
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Zou LK Wang HN Pan X Tian GB Xie ZW Wu Q Chen H Xie T Yang ZR 《Journal of Zhejiang University. Science. B》2008,9(7):536-545
The phyAm gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyAm-phyCs gene was successfully overexpressed in P. pastoris as an active and ex-tracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5～6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyAm. 相似文献
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Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice 总被引：1，自引：1，他引：0
Qing ZHANG Hui YU Feng-zhen ZHANG Zhi-cheng SHEN 《Journal of Zhejiang University. Science. B》2013,14(10):867-874
Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value.This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds,and a simple process for recovery and purification of rHSA for economical manufacture.An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference(RNAi) cassette suppressing the CYP81A6 gene.This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) cassette which confers glyphosate tolerance.ANX Sepharose Fast Flow(ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance(Butyl HP) hydrophobic interaction chromatography was used to purify rHSA.A transgenic rice line,HSA-84,was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds.This line also demonstrated high sensitivity to bentazon,and thus could be killed selectively by a spray of bentazon.A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%.Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA.This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism. 相似文献
18.
赵丹华 《广东教育学院学报》2011,(5):51-55
研究有机染料茜素红S（alizarin red S,ARS）与牛血清白蛋白（bovine serum albumin,BSA）、人血清白蛋白（human serum albumin,HSA）的结合反应,选择实验的最佳条件：PH=5．10的B—R缓冲溶液3．00mL,4．00mL5．0×10-4mol／L茜素红S溶液．反应20min后,体系的吸光度很稳定,在入λ=420nm处有最大吸收峰,并且随着牛血清白蛋白（BSA）,人血清白蛋白（HSA）的加入,茜素红S的吸收峰下降．因此以茜素红S为标记物,根据其在波长420nm处吸收峰下降的程度,可用于定量测定BSA和HSA．测量BSA的线性响应范围为0～32．0μg／mL,相关系数为0．9987,测量BSA的线性响应范围为0～28．0μg／mL,相关系数为0．9968．该方法具有较高的灵敏度和选择性,用于实际试样分析,实验结果令人满意． 相似文献
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双特异性磷酸酶（Dual Specificity Phosphatase）是酪氨酸磷酸酶家族中的一员,这个家族中的成员既能对磷酸化酪氨酸去磷酸化,也能对磷酸化丝／苏氨酸去磷酸化．此外,它们还在有丝分裂原激活蛋白磷酸酶信号途径（MAPK）中起重要生理功能．为了制备抗人双特异性磷酸酶的单克隆抗体（mAb）,以DUSP18重组蛋白为抗原免疫BALB／e小鼠,通过B淋巴细胞杂交瘤技术制备抗相应磷酸酶的mAb．用Western blot检测mAb对重组蛋白和细胞中天然磷酸酶的反应性,共获得6株可稳定分泌抗磷酸酶mAb的杂交瘤细胞株,为进一步研究双特异性磷酸酶的去磷酸化作用机理以及其在有丝分裂原激活蛋白磷酸酶信号途径（MAPK）中的信号转导提供强有力的工具． 相似文献
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采用PCR方法扩增对虾白斑病毒核糖核苷酸还原酶基因,插入到pGEX-4T-2表达载体上构建出带有目的基因的重组质粒pGEX-4T-2RR,然后将重组质粒转化大肠杆菌.转化菌经IPTG诱导后大量表达重组蛋白,通过降低诱导温度获得可溶性表达的重组蛋白,采用Glutathione Sepharose 4B亲和层析对重组蛋白进行纯化,获得了高纯度的目的蛋白. 相似文献