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1.
人睫状神经营养因子的原核表达,纯化及其生物效应   总被引:2,自引:0,他引:2  
人睫状神经营养因子(hCNTF)克隆入pBV220中,在DH5α菌株中表达,重组蛋白以包含体的形式存在,表达量为菌体总蛋白的50%左右。经比较发现用2mol/L脲洗涤包含体可溶解大量可溶性细菌蛋白,且包含体损失较小。在高浓度变性剂条件下进行sepharcylS-200凝胶过滤,解决了纯化中hCNTF易聚合的问题,在低浓度变性剂条件下进行DEAE离子交换,有利于蛋白活性的保持。经两步纯化后得到均一性hCNTF,纯度达95%以上。在自然状态下使hCNTF复性。纯化复性后的hCNTF对无血清培养的鸡胚背根节神经元和脊髓腹角运动神经元有明显的维持存活和促进生长发育的生物效应。  相似文献   

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人GM—CSF cDNA的克隆和在大肠杆菌中的表达   总被引:3,自引:0,他引:3  
从诱导的人胚肺细胞HFL株中提取总RNA.经RT-PCR反应获取了人GM-CSFcDNA,DNA序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用PCR改造了人GM-CSF的cDNA5’端核苷酸序列,并将改造的人GM-CSF基因插入含T7启动子的质粒pET-11d构建成表达质粒pETC-5,将此质粒转化大肠杆菌株BL21(DE3)得到表达菌株BLEC4。表达菌株用0.5mol/LIPTG诱导2小时后,产生大量重组蛋白并形成包涵体。SDS—PAGE电泳图谱扫描结果表明,rhGM-CSF产量占菌体总蛋白量的16%。ELISA和TF-1细胞培养测定表明,初步纯化和复性的rhGM-CSF具有天然的hGM-CSF生物活性。  相似文献   

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睫状神经营养因子对NO引起海马神经元毒性反应的影响   总被引:2,自引:0,他引:2  
Chen XQ  Chen ZY  Lu CL  He C  Wang CH  Bao X 《生理学报》1999,51(5):501-507
本研究采用原代培养大鼠海马神经元,观察睫状神经营养因子(ciliary neurotrophic factor,CNTF)对NO引起细胞毒性反应的影响。NO供体硝普钠与S-亚硝基-乙酰青霉胺,NOS底物L-Arg及钙载体ionomycin,均可引起海马神经元存活率下降,LDH漏出增加;提前24h给予不同浓度CNTF,均能提高神经元的存活率,减少LDH漏出,其作用呈剂量依赖性。  相似文献   

4.
为了提高人睫状神经营养因子(CNTF)的生物学活性,用PCR方法获取N端缺失14个氨基酸的CNTF基因片段,经酶切鉴定、核酸测序证实突变体的核苷酸序列,将其重组至表达质粒pBV220,构建了CNTF突变体表达载体pBV-CNTFΔ.用SDS-PAGE测定其表达水平,鸡胚背根节无血清培养法检测表达蛋白的生物学活性.结果表明,pBV-CNTFΔ能表达生物学活性高于天然CNTF的约26kD蛋白质,表达水平达30%.为今后通过基因工程方法获得CNTF突变体,从而制备高效的CNTF制剂创造了条件.  相似文献   

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人粒细胞集落刺激因子hG—CSF cDNA在大肠杆菌中表…   总被引:6,自引:0,他引:6  
在不改变编码蛋白质氨基酸序列的前提下,利用合成DNA接头的方法,在原始cDNA克隆基础上,构建了5'端密码子富含AT的hG-CSF cDNA突变体,使hG-CSF cDNA得以在大肠杆菌中表达,但表达水平很低,借助相同的手段,在hG-CSF cDNA 5'端增加24核苷酸对的FLAG肽编码序列,构建了hG-CSF杂合蛋白(在hG-CSF成熟蛋白N末端增加8氨基酸残基FLAG肽,二者结合点为肠激肽酶  相似文献   

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利用人粒细胞集落刺激因子(hG-CSF()cDNA3'端非翻译区(3'UTR)中存在的DraI酶切位点,通过部分酶切与完全酶切,删除3'-UTR不同长度构建了四种hG-CSF cDNA瞬时重组表达质粒,转染COS-7细胞手,生物活性测定结果提示,hG-CSFcDNA3'-UTR对其表达起负调控作用,其关键性序列位于紧接终止密码子TGA下游的65bp范围内,3'-UTR对hG-CSF cDNA表达的  相似文献   

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利用人粒细胞集落刺激因子(hG-CSF)cDNA3′端非翻译区(3′-UTR)中存在的DraⅠ酶切位点,通过部分酶切与完全酶切,删除3′-UTR不同长度,构建了四种hG-CSFcDNA瞬时重组表达质粒。转染COS-7细胞后,生物活性测定结果提示,hG-CSFcDNA3′-UTR对其表达起负调控作用,其关键性序列位于紧接终止密码子TGA下游的65bp范围内,3′-UTR对hG-CSFcDNA表达的影响与转录水平的差别有一定关系。  相似文献   

8.
人铜锌超氧化物歧化酶cDNA的克隆,测序及表达   总被引:17,自引:1,他引:16  
用逆转录聚合酶链反应(RTPCR),以人胎肝组织总RNA为模板,扩增了人铜锌超氧化物歧化酶(hCu,ZnSOD)的cDNA,并进行序列分析,将该hCu,ZnSODcDNA重组到T7启动子控制下的分泌型表达载体pET22b(+)中,构建表达质粒pETSOD,并转化大肠杆菌BL21(DE3)。SDSPAGE及蛋白质印迹分析表明,经1mmol/L异丙基硫代βD半乳糖苷(IPTG)诱导后,可高效表达一分子量为19kD的蛋白质,与抗人SOD多抗有特异的免疫反应,表达量约为菌体总蛋白质的30%,具有特异性SOD酶活性,酶活力可达1797u/ml培基。  相似文献   

9.
重组人干细胞因子   总被引:2,自引:0,他引:2       下载免费PDF全文
选择大肠杆菌的偏性密码,合成了可溶形式的人干细胞因子(hSCF)的cDNA。通过PCR完成了合成片段的一次性组装与克隆,并在E.coli中进行了温控型的高效表达,目的蛋白可占菌体总蛋白的40%左右。表达产物复性后,经离子交换、凝胶过滤层析,得到了电泳纯的rhSCF。经测定,rhSCF氨基端序列及其它理化性质与天然hSCF完全一致,并可刺激人骨髓细胞的增殖,导致粒、巨核系集落(CFU-GM)大小、数量的明显增加,显示天然hSCF的生物活性。  相似文献   

10.
大鼠卵巢绒毛膜促性腺激素受体在CHO中的表达和扩增   总被引:1,自引:0,他引:1  
本文报道了利用二氢叶酸还原酶放大系统将大鼠LH/hCG受体(记为F-hCGR)及其胞外肽段(记为T-hCGR)在中国苍鼠卵巢细胞(CHO)中的表达。SDS-PAGE分析表明,F-hCGR为一条蛋白质带,其表观分子量为92kd,而T-hCGR为35kd和37kd两条带。表达受体对其配基hCG表现出高的亲合力,F-hCGR的解离常数为7×10-9mol/L,T-hCGR为6.4×10-9mol/L。表达F-hCGR的转染CHO细胞可结合125I-hCG,而表达T-hCGB者不结合125I-hCG。这提示F-hCGR主要存在于细胞质膜表面上。表达F-hCGR的转染CHO细胞能刺激。cAMP的形成,而表达T-hCGR者不能刺激cAMP形成。免疫荧光定位结果表明,T-hCGR主要分布于质膜的细胞质侧以及胞内其他一些细胞器膜上。用免疫亲和层析可以得到纯化的T-hCGR。  相似文献   

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Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.  相似文献   

16.
Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.  相似文献   

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Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.  相似文献   

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正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases  相似文献   

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