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The present invention relates to a process of producing a functional immunoglobulin superfamily protein, which has at least one disulphide bond when functional, the process comprising the steps of providing a bacterial cell comprising a gene coding for the protein, the gene is expressible in said cell, cultivating the cell under conditions where the gene is expressed, isolating the protein from the cell without reducing it, and subjecting the isolated protein to a folding treatment.

本发明涉及一种产生功能性免疫球蛋白超家族蛋白的方法,此蛋白质在有功能时具有至少一个二硫键,此方法包括如下步骤:提供一种包含编码此蛋白质的基因且此基因可在其中表达的细菌细胞;在使此基因表达的条件下培养此细胞;从该细胞中分离出此蛋白质而不还原它;以及使分离出的蛋白质经受折叠处理。

With the aid of PDQuest 2-DE software, 11 significant protein dots were found out and then 9 of them are pick out to go through peptide mass fingerprinting based on MALDI TOF MS and NCBI database searching . In all, 5 proteins were identified: Ribulose bisphosphate carboxylase , Nucleic acid-binding protein , Ribosomal protection tetracycline resistance protein , hypothetical protein FG04726.1 [Gibberella zeae PH-1], nitric oxide synthase .

应用PMF技术对这11个具有显著表达差异蛋白点中的9个进行鉴定,得到了5个质谱阳性鉴定结果,其中2个蛋白分别为玉米种属的1,5-二磷酸核酮糖羧化酶/加氧酶和叶绿体的核酸结合蛋白;其它的3个蛋白在玉米蛋白质数据库中未见报道,但与其它物种基因编码的蛋白质具有高度的序列同源性。

Nucleoprotein is also a major immunogenetic antigen for SARS; however, several tests have shown that N protein had the reactivity with other coronavirus positive sera. It is therefore an advantage of S protein as recombinant target antigen for SARS serum diagnosis compared with recombinant N protein. Moreover, S protein has been shown to be the major antigenic determinant that induces neutralizing antibodies and protective immunity to SARS-CoV, so it is also important in designing SARS genetic engineering vaccine.

SARS-CoV S 蛋白由1255 个氨基酸组成,体外表达如此大的蛋白很困难,而且表达蛋白的可溶性不高;其次,实验已经证实,全长S 蛋白含有大量无关的表位,致使表达的全长S蛋白对SARS 阳性血清的检出率低于S 蛋白片段;另外作为疫苗研究,S 蛋白上大量无关表位作为异体蛋白进入体内,会引发机体的过度免疫反应,这也是SARS-CoV 可能的致病机理之一。

Results We obtained a lysogenic bacteriophage MZTP01with clear plaque and 1 mm diameter. Fragment D with 2362bp (Genebank No. AY639599) was obtained after the phage DNA hydrolyzed by HindⅢ/EcoRⅠ. Among fragment D, the pep gene with molecular weight of 47 kDa and length of 1101bp was cloned and expressed. Recombinant M15 (pQE30pep) was built and overexpressed in Escherichia coli with a 47kDa clear band. At the same place a clear band was observed by Western blot. Judging from the time course expression, we could conclude that PEP protein produced at 1 hour after induction and then increased gradually. PEP protein was mainly in the form of inclusion body in the recombinant and slowed the growth speed of host. Homologous comparison of PEP protein from phage MZTP01 with other PEPs from BLAST were that phage MZTP01 PEP protein had 100% homologe with that of Escherichia coli K12, and most of others took the similarity in the range between 37%~84%.

诱导获得的溶原性噬菌体MZTP01斑点清晰,直径约1mm,成斑时间12h;从噬菌体基因组DNA双酶切(HindⅢ/EcoRⅠ)片段中回收长度为2362bp的D片段(Genbank登录号: AY639599),又从D片段中克隆了长度为1101bp、编码367aa、分子量为47kDa的pep基因,表达载体M15(pQE30pep)在大肠杆菌(Escherichia coli, E.coli)中表达获得了47kDa的清晰表达带,在1h 时开始产生蛋白并有逐步上升的趋势; Western blot 也在47kDa处得到一条清晰的条带;可溶性分析表明PEP蛋白在重组菌株中是以不可溶的包含体形式存在的,该蛋白的产生明显地抑制了宿主的生长速度;噬菌体PEP氨基酸序列之间的同源性比较表明,噬菌体MZTP01 PEP蛋白与来自E.coli K12噬菌体的PEP蛋白的同源性程度最大。

In accordance with the invention, the hG-CSF protein can be prepared with high purity through rather simple process facilitating secretion of large amount of hG-CSF fusion protein into the periplasm, which does not require complicated processes such as solubilization and subsequent refolding required for isolation of the hG-CSF protein produced in cytoplasm as insoluble inclusion bodies by conventional techniques, thus, the hG-CSF protein can be widely used as an active ingredient in the development of supplementary agents for anticancer therapy.

根据本发明,通过促进大量hG-CSF融合蛋白分泌到周质中的相当简单的方法可以高纯度制备hG-CSF蛋白,所述方法不需要复杂的加工过程例如对于通过常规技术分离胞质中产生的作为不溶性包函体的hG-CSF所需的溶解和随后再折叠,因此,在开发用于抗癌治疗的补充药物的过程中可用hG-CSF蛋白广泛地作为活性成分。

Preliminary Study on Enzymatic Modification of Protein──Study on Plastein Reaction of Soybean Protein with Sesame Protein;2. Utilizing alkaline hydrolysate of rice protein as substrate,the effect of plastein reaction condition on the yield of plastein was studied.

以大米蛋白的碱性蛋白酶水解产物为原料,研究了反应条件对类蛋白反应产率的影响,采用正交试验优化了类蛋白反应的条件,并分析了最佳条件下类蛋白的氨基酸组成。

The inhibitory effect of different component from the filtrate of two antagonists was tested: the polysaccharose, crude fat, and protein produced by Gvirens had inhibitory activity to the spore germination of P.funerea, and the crude fat and protein was more significant, the protein more stable. The crude fat from B.firmus could completely inhibit the Rfunerea's spore germination, the inhibitory effect of polysaccharose and protein also was stable, but polysaccharose more excellent.

G.virens与B.firmus的亲合性研究显示,G.virens发酵滤液在70%浓度以下对B.firmus的生长繁殖无明显抑制作用,在70%以上浓度,B.firmus的生物量有所下降;而B.firmus发酵滤液对G.virens的生长繁殖无明显抑制作用,这种状况有利于二者在植物病害生物防治中协同作用的发挥,表明了两种微生物在松赤枯病生物防治中联合作用的可能性。

The results of protein synthesis of NT embryos indicated that the protein synthesis pattern of NT embryos and fertilized eggs was similar. 19. 2 KD protein existed in 32-cell stage donor embryos disappeared in NT embryos at the pronuclear stage, however, this protein was reprogrammed to reappeared in 32-cell stage NT embryos.

七、核移植胚的蛋白合成分析结果表明,原核期核移植胚与原核期受精卵具有相同的合成模式。32-细胞期供体胚中的19.5KD蛋白在原核期重组胚中消失,而在32-细胞期重组胚中再度出现,发生了蛋白合成的重序过程。

The rice reductional division gene coded protein is the protein having SEQ ID No.2 amino-acid residue of the sequence table, or it is the protein derived from SEQ ID No.2, having the activity same as the sequence of the SEQ ID No.2 amino-acid residue, in which protein, SEQ ID No.

水稻减数分裂基因编码的蛋白,是具有序列表中SEQ ID №:2氨基酸残基序列的蛋白质,或者是将SEQ ID №:2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与SEQ ID №:2的氨基酸残基序列相同活性的由SEQ ID №:2衍生的蛋白质。

The protein OsSPO11-1 coded with rice reductional division gene OsSPO11-1 is the protein having SEQ ID No.2 amino-acid residue of the sequence table, or, it is the protein derived from SEQ ID No.2 in which protein, SEQ ID No.2 amino acid residue sequence being proceeding substitution, deletion or addition with one or more amino acid residue, but having the same activity of SEQ ID No.2 amino acid residue sequence.

水稻减数分裂基因OsSPO11-1编码蛋白OsSPO11-1,是具有序列表中SEQ ID №:2氨基酸残基序列的蛋白质,或者是将SEQ ID №:2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与SEQ ID №:2的氨基酸残基序列相同活性的由SEQ ID №:2衍生的蛋白质。

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