人乳铁蛋白基因在烟草中的表达及抗细菌与病毒的能力

从人血液中性白细胞中分离细胞总ＲＮＡ,用ｏｌｉｇｏ（ｄＴ） 为引物进行ｃＤＮＡ 第１ 条链合成,再用乳铁蛋白ｃＤＮＡ 的特异性引物进行聚合酶链反应（ＰＣＲ） ,分两段合成并拼成完整的乳铁蛋白ｃＤＮＡ。核苷酸序列测定证明,该ｃＤＮＡ 所编码的氨基酸与前人论证的人乳铁蛋白的氨基酸序列完全一致。将该ｃＤＮＡ 构建成植物表达载体ｐ３５ＳｈＬＦｃ,经根癌农杆菌ＬＢＡ４４０４ 感染烟草叶盘,获得具有卡那霉素抗性的烟草植株,ＰＣＲ检测和Ｓｏｕｔｈｅｒｎ杂交表明,人乳铁蛋白基因已整合到烟草基因组中。Ｎｏｒｔｈｅｒｎ 杂交与Ｗｅｓｔｅｒｎ 免疫印迹分析,检测到人乳铁蛋白基因在转化烟草中表达。体外实验证明,人乳铁蛋白基因在烟草中的表达产物对植物致病菌烟草火叶病菌、水稻白叶枯病菌有一定抑制作用     

人乳铁蛋白基因克隆及细胞表达研究

通过PCR法直接克隆了2.366kb的人乳铁蛋白基因cDNA序列及800bp的山羊β乳球蛋白基因5′ 端调控序列,并连接到表达载体pLNCX中。利用脂质体包裹含人乳铁蛋白基因cDNA的重组质粒pLNCXHLF,并导入到小鼠乳腺癌细胞株MA∕782中,G418及PCR筛选获得阳性单克隆细胞,增殖后,转染细胞利用海藻酸钠固定化包埋培养,经激素诱导,培养液上清通过Western印记检测证明,转染细胞表达并分泌出人乳铁蛋白,分子质量为34kDa;ELISA法测出,每升培养基(含105个细胞)重组蛋白最高表达量为65mg;抗菌实验表明,所获得的重组人乳铁蛋白具有抑制大肠杆菌生长的作用,而且比人乳铁蛋白标准品作用更强。 Abstract:In this paper,we directly cloned 2.366Kb cDNA sequence of human lactoferrin gene and 800bp 5′flank regulatory sequence of β/lactoglobulin gene from goat by PCR,then connected them with the expression vector pLNCX.The recombinant plasmid pLNCXHLF containing human lactoferrin gene cDNA was transfected into mice mammary tumor cell line MA/782 after liposome transinfection.Positive single clone cells were selected with G418 and by PCR.After proliferating,the transfected cells immobilized and cultured in soldium alginate were induced by hormone.The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLF protein,whose MW is 34 KD.The highest amount detected by ELISA reached 65mg/l medium/105 cells.The result of antibacterial experiment indicates that the recombinant hLF protein has the effect of inhibiting E.coli proliferation;moreover,its activity is superior to the commercial available hLF′s.     

人GDNF基因在昆虫细胞中的高效表达

应用昆虫杆状病毒表达系统在昆虫细胞Ｔｎ－５Ｂ１－４中高效表达了人胶质细胞源性神经营养因子（ＧＤＮＦ）,ＰＡＧＥ分析表达量占细胞可溶性蛋白质的３０％左右,表达产物经亲和层析纯化后纯度达８０％以上,活性研究表明,昆虫细胞表达的ＧＤＮＦ蛋白能显著促进多巴胺能神经元的存活,此研究为进一步研究ＧＤＮＦ结构与功能打下了良好的基础。     

人乳铁蛋白cDNA的克隆及序列分析

从北京正常人乳腺组织中提取总RNA,用RT-PCR的方法扩增人乳铁蛋白(hLF)的cDNA,将其克隆到pGEM-T载体上并进行DNA序列测定。结果表明,所克隆的hLF cDNA序列全长为2136bp,其DNA序列与GenBank中另外5个hLF cDNA序列相比,有2个碱基与这5个序列不同：1740位这5个序列是G,本文序列是C;1756位这5个序列是T,本文序列是C。其中1740位碱基的变化导致了第580位氨基酸由Glu变为Asp。     

杆状病毒载体及其在昆虫细胞中表达外源基因的研究进展

本文介绍了杆状病毒载体在昆虫细胞中表达外源基因的基本策略和发展趋势。杆状病毒载体系统近年来已被人们广泛用来表达人类、动物和植物等的一些重要蛋白质分子,在医学、农业等领域的基因工程研究中发挥了越来越大的作用。杆状病毒载体系统表达外源基因的效率高,表达产物的结构和活性与天然产物一致,为当今基因工程研究中最有发展前途的病毒载体表达系统。     

外源基因在昆虫杆状病毒表达系统中的表达

随着杆状病毒载体和筛选方法的不断改进,通过Bac-to-Bac方法可以使杆状病毒最大重组率达到100%,缩短了构建重组载体的时间,极大提高了工作效率。另外,研究者开发了一些新的宿主域扩大的昆虫杆状病毒载体,能够在家蚕或蛹内进行高水平表达重组蛋白。昆虫杆状病毒表达系统具有完备的翻译后加工修饰功能和高效表达外源蛋白的能力等特点,是一种非常理想的真核表达系统。利用该表达系统现已成功表达了约千种外源蛋白。以重组杆状病毒为载体的昆虫表达系统、外源基因在该表达系统中的表达情况及在农业领域中的应用进行了介绍。     

人乳铁蛋白在转基因马铃薯块茎中的表达

人乳铁蛋白(human lactoferrin,hLF)是人体非特异性免疫系统的重要成员之一,具有抗细菌、真菌和抗病毒活性及其他多种功能.报道将hLF基因的cDNA与马铃薯(Solarium tuberosum L.)块茎专一性表达patain基因启动子融合后通过农杆菌介导导入马铃薯,PCR检测证实获得了多个转基因株系,RT-PCR阳性结果说明hLF mRNA在马铃薯植株中得到了表达.同时,经过ELISA及Western blot检测证实,转基因马铃薯表达了hLF并具有人乳铁蛋白的活性.     

丙型肝炎病毒NS2-3蛋白酶基因在Sf9昆虫细胞中的表达

丙型肝炎病毒(hepatitis C virus,HCV)为单股正链RNA病毒,其基因组长约9.5kb,5'端和3'端各有一个长约345bp和60bp的非编码区,编码区含一个大开放读码框架,编码3 010aa～3 033aa残基的多蛋白前体.     

人尿激酶原cDNA在昆虫杆状病毒真核表达系统中的高效表达

人尿激酶原是一种新型溶栓剂,优于尿激酶,具有血纤维蛋白特异性,为了在昆虫杆状病毒表达系统中高效表达ｐｒｏ－ＵＫ,我们在ｐＡｃ３７３基础上,插入野生型ＡｃＭＮＰＶ ｐｏｌｙｈｅｄｒｉｎ启动子区－７～－１碱基序列,构建了一个高表达转移载体ｐＡｃＹＴ．分别经三次克隆将ｐｒｏ－ＵＫ ｃＤＮＡ正向插入到转移载体ｐＡｃ３７３或ｐＡｃＹＴ的ＢａｍＨＩ－ＫｐｎＩ位点上。用Ｌｉｐｏｆｅｃｔｉｎ将ｐＡｃＹＴ－ＵＫＤＮ     

邻苯二酚2,3-双加氧酶的结构和功能研究进展

邻苯二酚是所有芳香族化合物降解过程中的重要的中间产物,其降解有邻位和间位裂解两条裂解途径,分别由邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)催化裂解。本综述简要介绍了邻苯二酚2,3-双加氧酶的结构和功能的研究进展。     

邻苯二酚2,3—双加氧酶在大肠杆菌的表达与定域

本文在大肠杆菌/枮草芽孢杆菌间的穿梭质粒pTG 402的基础上构建了几个新的带有显色标志基闲xylE的表达质粒,摸索了该基因所编码的邻苯二酚2,3-双加氧酶(CatO_2ase)的表达条件,分析了该酶一级结构与二级结构的亲水性和疏水性,测定了它在大肠杆菌中的产量与分布。结果表明,CatO_2ase与各质粒的表达量不等,表达量高低与培养时间、宿主菌及诱导与否等影响因素有关;表达后有部分酶可在胞外测出,但大部分仍定域于膜内,亲、疏水性分析示该酶不具分泌性蛋白的显著特点。因该酶易于检测和定量,可作为一种选择性标记和监测指示系统在基因工程中推广应用,同时亦为用基因工程菌消除芳烃类化合物的污染提供了理论依据。     

洋葱伯克霍尔德氏菌产邻苯二酚2,3-双加氧酶的研究

对洋葱伯克霍尔德氏菌L 68生长及产邻苯二酚2,3 双加氧酶(C23D)的条件进行了研究,其最适产酶pH7.2;最适生长温度30～35℃;最适培养时间48h;苯酚浓度0.09%最有利于菌体产酶.对菌株L 68产生的C23D酶进行了纯化,超声波破碎后的细胞提取液经硫酸铵分级沉淀、DEAESepharoseFastFlow层析、Hydroxyapatite层析、SephadexG 150层析后,收率为20%,酶比活力提高了230倍.SDS PAGE检测得到了分子量为(34±1)kDa的蛋白.     

Catechol 2,3-dioxygenase from the thermophilic, phenol-degrading Bacillus thermoleovorans strain A2 has unexpected low thermal stability

Catechol 2,3-dioxygenase from the thermophilic Bacillus thermoleovorans A2 was purified and characterized. The catechol 2,3-dioxygenase has a molecular mass of 135 000 Da and consists of four identical subunits of 34 700 Da. One iron per enzyme subunit was detected using atom absorption spectroscopy. Enzyme activity was not inhibited by EDTA, suggesting that the iron is tightly bound. Addition of hydrogen peroxide to the enzyme completely destroyed activity, indicating that the iron was in the divalent state. The isoelectric point of the enzyme was 4.8. The enzyme displayed optimal activity at pH 7.2 and 70°C. The half-life of the catechol 2,3-dioxygenase at the optimum temperature was 1.5 min under aerobic conditions and 10 min in a nitrogen atmosphere. This stability of the enzyme is comparable to the stability of the enzyme from the mesophilic Pseudomonas putida mt-2. The stability of the cloned enzyme in E. coli extracts was identical to the stability in wild-type extracts, suggesting that no stabilizing factors were present in Bacillus thermoleovorans A2 In whole cells the half-life of the enzyme at 70°C was approximately 26 min, when protein synthesis was disrupted by chloramphenicol; however, the activity remained constant when protein synthesis was not inhibited. From these results we concluded that catechol 2,3-dioxygenase from Bacillus thermoleovorans A2 is not particularly thermostable, but that the organism retains the ability to degrade phenol at high temperatures because of continuous production of this enzyme. Received: October 10, 1998 / Accepted: March 18, 1999     

菠菜光系统Ⅱ天线组分CP29的分离及其性质

菠菜放氧的光系统Ⅱ（ＰＳⅡ）核心复合物经０．８ｍｏｌ／Ｌ Ｔｒｉｓ（ｐＨ８．０）洗涤后,用温和的非离子去垢剂ＤＭ和高浓度的ＬｉＣｌＯ４增溶,再经ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ－６５０Ｓ离子交换柱层析分离,可得到ＰＳⅡ天线组分中的叶绿素α／ｂ结合蛋白（ＣＰ２９）。ＳＤＳ－ＰＡＧＥ显示一条３０ｋＤ蛋白质带。根据Ａｒｎｏｎ法和Ｍａｒｋｗｅｌｌ法的结果表明,每个蛋白质分子结合有７～８个分子的叶绿素α和２～３     

Indoleamine 2,3 dioxygenase and regulation of T cell immunity

Regulation of adaptive immune responses is critically important to allow the adaptive immune system to eradicate infections while causing minimal collateral damage to infected tissues, as well as preventing autoimmune disease mediated by self-reactive lymphocytes. Tumors and pathogens that cause persistent infections can subvert immunoregulatory processes to protect themselves from destruction by T cells, to the detriment of patients. A growing body of evidence supports the hypothesis that specialized subsets of dendritic cells expressing indoleamine 2,3 dioxygenase (IDO), which catalyzes oxidative catabolism of tryptophan, play critical roles in regulation of T cell-mediated immune responses. IDO-dependent T cell suppression by dendritic cells suggests that biochemical changes due to tryptophan catabolism have profound effects on T cell proliferation, differentiation, effector functions, and viability. This has critical implications for immunotherapeutic manipulations designed for patients with cancer and chronic infectious diseases. In this review, I focus on dendritic cells that can express IDO, and which acquire potent T cell regulatory functions as a consequence.     

Bifidobacteria alleviate experimentally induced colitis by upregulating indoleamine 2, 3‐dioxygenase expression

The goal of this study was explore the role of indoleamine 2, 3‐dioxygenase (IDO) in the therapeutic effect of probiotics on inflammatory bowel disease (IBD). Trinitrobenzene sulfonic acid (TNBS) was used to induce colitis in mice and 1‐methyltryptophan (1‐MT) to block expression of IDO. Clinical manifestations and macroscopic and microscopic colonic changes were assessed using a disease activity index (DAI), the Wallace–Keenan, and Curtner scoring systems, respectively. Expression of colonic IDO was detected by western blot. Immunohistochemistry analysis to evaluate numbers of CD11c⁺ cells and expression of IL‐17 and Foxp3 showed that DAI, Wallace–Keenan, and Curtner scores were lower in the Bifidobacteria treatment group than the control group and that the therapeutic effect of Bifidobacteria was blocked by 1‐MT (P < 0.05). Additionally, Bifidobacteria were found to increase expression of IDO and the numbers of CD11c⁺ cells, CD11c⁺ and IDO double positive cells and Foxp3⁺ Treg cells, while decreasing the number of IL‐17⁺cells (P < 0.05). The generation of Foxp3⁺ Treg cells induced by Bifidobacteria was abrogated by 1‐MT (P < 0.05). These findings study suggest that Bifidobacteria attenuate TNBS‐induced colitis by inducing expression of IDO, which further increases generation of Foxp3⁺ Treg cells.

     

Structural and functional analyses of human tryptophan 2,3‐dioxygenase

Tryptophan 2,3‐dioxygenase (TDO), one of the two key enzymes in the kynurenine pathway, catalyzes the indole ring cleavage at the C2‐C3 bond of l ‐tryptophan. This is a rate‐limiting step in the regulation of tryptophan concentration in vivo, and is thus important in drug discovery for cancer and immune diseases. Here, we report the crystal structure of human TDO (hTDO) without the heme cofactor to 2.90 Å resolution. The overall fold and the tertiary assembly of hTDO into a tetramer, as well as the active site architecture, are well conserved and similar to the structures of known orthologues. Kinetic and mutational studies confirmed that eight residues play critical roles in l ‐tryptophan oxidation. Proteins 2014; 82:3210–3216. © 2014 Wiley Periodicals, Inc.     

Optimization of a Thermostable Lipase from Bacillus stearothermophilus P1: Overexpression, Purification, and Characterization

An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55°C. It was highly stable in the temperature range of 30–65°C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37°C in 0.1% Chaps and Triton X-100.