具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化

用PCR法从质粒pHB3中扩增了人红细胞带3蛋白胞质片段(CDB3)基因.PCR产物经限制性内切酶切割后与多克隆位点处带有编码6个组氨酸序列的高效表达载体pET28b连接,构建为重组子pCDBHistag.重组子经酶切及序列测定后在大肠杆菌BL21(DE3)中获得高效表达,可溶性目的蛋白占菌体总蛋白的40%左右.C端带有6个连续组氨酸的带3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度,而且简化了目的蛋白的纯化过程.经一步螯合Ni²⁺的亲和层析获得了电泳纯的带3蛋白胞质片段融合蛋白.活性测定结果表明,带3蛋白胞质片段融合蛋白能够抑制醛缩酶(Aldolase)活性的70%,与文献报道的人红细胞内带3蛋白胞质片段具有相同的功能.     

金属硫蛋白家族内的结构域拼接

金属硫蛋白(Metallothioneins,MTs)由结构独立且功能明显区别的β,α两个结构域组成。神经生长抑制因子(NeuronalGrowthInhibitoryFactor,GIF)双名金属硫蛋白-Ⅲ(MT-Ⅲ),是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白,而β－结构域为其功能结构域。为深入系统地研究MTs,尤其是GIF及其结构域的结构与功能,我们构建了金属硫蛋白家族内结构域拼接体βGIF-αMT-1(βⅢ-αⅠ)和βMT-1-αGIF(βⅠ-αⅢ):PCR扩增得各个结构域的cDNA序列,酶切后克隆入原核表达载体pGEX-4T-1,经发酵、诱导表达、亲和层析、凝血酶切和进一步纯化,得率约为80mg蛋白/L菌液。测其电泳行为、氨基酸组成、质谱、金属巯基含量等,证明得到了目的蛋白。紫外吸收图谱和圆二色性图谱显示,结构域拼接体拥有金属硫蛋白家族成员的特征金属巯基簇结构域,初步功能实验表明,βⅢ－αⅠ也具有抑制神经元生长的功能。     

模拟胃肠道消化液中小球藻类金属硫蛋白对镉离子的结合作用研究

利用锌诱导小球藻,通过提取分离和纯化,获得锌结合类金属硫蛋白(MT-like蛋白).通过透析过程,研究模拟胃、肠道消化液中MT-like蛋白对Cd2+的结合作用.结果表明,在模拟胃液中MT-like蛋白处于脱金属状态,与Cd2+的结合能力弱.而在肠消化液中,MT-like蛋白能与Cd2+结合,20 mg·L-1 MT-...     

低分子量紫色酸性磷酸酶多克隆抗体的制备及应用

将最近从地瓜中提出来的低分子量紫色酸性磷酸酶（smPAP)基因克隆到GST融合蛋白表达载体pGEX－2T中，在大肠杆菌BL21codon plus中进行表达，用表达的融合蛋白免疫兔子产生多克隆抗体，用抗血清在昆虫细胞中表达的smPAP和地瓜提取液中分离纯化的PAP进行检测，产生了良好的交叉反应。     

HIV-1嵌合抗原的纯化及免疫原性分析

为获得高效的HIV诊断试剂,选定HIV-1的外膜蛋白env中469-511aa,538-674aa和700-734aa3处包含较多抗原位点的区域作为免疫抗原,用PCR的方法从HIV-1全基因扩增编码这3处片段的基因序列,将它们克隆到原核表达载体中,利用大肠杆菌表达系统表达嵌合蛋白.结果发现,3段嵌合基因能在大肠杆菌BL21(Star)中表达,通过Ni-sepharose4B金属Ni螯合层析柱分离纯化目的产物,酶联免疫检测结果表明,纯化抗原有较强的抗原特异性.     

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.     

用HpLC反相柱层析纯化玉米根镉结合蛋白

本研究建立了纯化玉米根镉结合蛋白的有效方法.其步骤为用加热处理由CdCl_2诱导形成的玉米根的粗提物,经过离子交换柱层析后的Cd结合复合物,连续用HpLC反相柱层析,获得了纯的蛋白.该蛋白质的分子量为3200D;半胱氨酸含量为29.6％;每个蛋白质分子结合大约3个Cd原子;Cd与半胱氨酸的比值为1:2.3.根据其特性说明它可能是植物类金属硫蛋白.     

膀胱癌分子标志物基质金属蛋白酶1的可溶性表达和尿液中的活性分析

通过分离人胚胎肾细胞(HEK293)中基质金属蛋白酶1(MMPl)基因序列,利用基因工程技术在大肠杆菌中表达并纯化了包涵体复性的MMP1;采用融合表达硫氧还蛋白(Thioredoxin,TrxA)的方法获得了可溶性的MMPl-TrxA复合蛋白,在TrxA蛋白和MMP1活性中心之间引入了一个肠激酶切割位点,通过酶切作用获得可溶性MMP1.明胶酶谱分析法对体外表达的MMP1和体内(尿液中)MMP1进行了分析比较.结果表明,TrxA蛋白能够显著提高MMP1的可溶性,且可溶性MMP1的明胶降解活性是复性的MMP1降解活性的1.54倍,与尿液中的MMP1的明胶酶降解活性相当.因此,明胶酶谱法是一种有效的、高灵敏的MMP1检测方法,所表达的可溶性MMP1为小分子探针筛选提供了更可靠的分子靶点.     

抗黄曲霉毒素B1多价纳米抗体融合蛋白的构建及活性分析

纳米抗体来源于天然缺失轻链的重链抗体可变区,是已知最小抗原结合单元.该研究构建了抗黄曲霉毒素B1(AFB1)纳米抗体的单价及多价串联体,分别与绿色荧光蛋白(GFP)编码片段融合并克隆至原核表达载体pET30.以大肠杆菌BL21(DE3)作为表达宿主,通过异丙基-β-D硫代吡喃半乳糖苷诱导,亲和层析技术分别纯化单、双及三...     

人野生型MBL基因在大肠杆菌中的表达

为获得人MBL蛋白，并对其功能进行初步研究，用DNA重组法构建了组氨到标签融合原核表达质粒pET28(b)-MBL。将重组质粒转入大肠杆菌BL21（DE3），经IPTG在37℃条件下诱导培养，利用SDS－PAGE，Westem-blot检测目的蛋白的表达，用IMAC金属螯合层析柱对其进行纯化。成功地表达了重组MBL蛋白，纯化的MBL浓度约为844μg/mL，为制备MBL的基因工程抗体奠定了基础。     

Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids

Affinity tags are efficient tools for protein purification. They allow simple one-step purification of proteins to high purity. However, in some cases the tags cause structural and functional changes in a protein, and need to be removed. Therefore, affinity tags that are readily introduced into proteins with minimal perturbation and have specific affinity for purification are desired. Herein, two metal-chelating amino acids derived from 2,2′-bipyridine and 8-hydroxyquinoline were genetically incorporated into glutathione S-transferase (GST) and the mutant proteins were purified by using the metal ion affinity of the unnatural amino acids. The purification of the GST mutants containing 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) showed that the proteins could be efficiently enriched in Ni–NTA by the metal ion affinity of the unnatural amino acid and purified to excellent purity. This method should be very useful for general protein affinity purification, especially for proteins whose structure or function is affected by affinity tags fused to N- or C-terminals.     

两步柱色谱法分离纯化重组肿瘤血管生长抑制因子Kringle 5

建立了一种用Ni2+螯合的Chelating Sepharose Fast Flow亲和柱色谱和Sephadex G-75凝胶排阻柱色谱分离纯化重组肿瘤血管生长抑制因子Kringle 5的方法。采用该工艺得到的重组Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明其纯度约为98%,且具有抑制鸡胚绒毛尿囊膜新生血管生成的生物活性。     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

灵芝发酵液中蛋白酶抑制剂GLPIA2的纯化及其特性

采用乙醇分级沉淀、凝胶色谱纯化、阴离子交换色谱分离等步骤从灵芝深层发酵液中提取得到蛋白酶抑制剂GLPIA1 与GLPIA2。其中GLPIA2仅在215 nm处有紫外吸收,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定为单一条带,相对分子质 量为15000。由其氨基酸组成分析谱图可看出,其酸性氨基酸含量较高,碱性氨基酸及芳香族氨基酸含量较低。GLPIA2抑 制剂的底物特异性研究表明,它对天冬氨酸族的胃蛋白酶和酵母蛋白酶A有相对较强的抑制作用。     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

离子交换色谱法分离纯化鸡卵黄免疫球蛋白

建立了高效、经济、大规模获得鸡卵黄免疫球蛋白(IgY)的生产方法。在对传统的水稀释法改良的基础上,结合聚乙二醇沉淀与离子交换色谱进行IgY的分离纯化。结果显示,用8倍无菌水稀释蛋黄液,用0.1 mol/L HCl调节pH为5.2,在4 ℃下静置8 h,于5000×g力离心可得上清粗IgY液,经测定回收率可达93.47%。然后用6%聚乙二醇沉淀后经DEAE-Toyopearl 650 M离子交换纯化,最佳的纯化条件: 0.05 mol/L磷酸盐缓冲液(PBS, pH 7)平衡上样,0.075 mol/L PBS(pH 7)洗脱。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果显示所得的IgY的纯度为95.02%,活性保持率高达73.77%。本研究弥补了传统分离方法不能同时达到高纯度和高回收率的缺点,且可用于大规模生产。     

An improved method for isolation and identification of Zn-metallothionein from cadmium-induced rat liver.

Zn-metallothioneins (MT-1 and MT-2) were isolated and purified from Wistar rat liver induced by subcutaneous injection with cadmium chloride over a short time. Instead of Sephadex G-50 and DEAE Sephadex A-50, new chromatographic media produced by Pharmacia, Sephacryl S-200, S-100 and DEAE Sepharose Fast Flow were used in the purification of metallothioneins. The time required for purification with the new method was only 1/3 that required with the usual method and had the same purification effect and rate of recovery. The number of mercapto groups measured with modified Ellman's reagent and cysteine as standard is 20 in MT molecules. Zn and Cd concentrations in each fraction were measured by single sweep polarography rather than atomic absorption spectrophotometry. MT-1 and MT-2 contained 6 gram atoms of zinc, but no cadmium. Purified MT-1 and MT-2 were shown by high performance liquid chromatographic analysis to be highly homogeneous and had an amino acid composition similar to that of Cd-MT.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

新型抗HIV-1蛋白GRFT的构建、表达及活性研究

根据已知的新型抗HIV-1蛋白GRFT基因氨基酸序列,推测其DNA编码序列,密码子优化及修饰后进行全基因化学合成,连接到原核表达载体pET28a（+）中,转化大肠杆菌BL21(DE3),IPTG诱导表达,获得目的蛋白. SDS-PAGE、Western Blot分析结果表明,目的蛋白得到良好表达并具有抗原活性;对表达条件进行优化,在最佳表达条件下,目的蛋白的表达量可占菌体总蛋白的55.84%;利用Ni2+-NTA柱亲和层析法进行目的蛋白的复性和纯化,凝胶成像系统扫描分析表明,纯度可达94.06%;运用Dot-ELISA法进行复性蛋白的抗原结合活性检测,结果显示,目的蛋白能够与HIV-1膜蛋白抗原特异性结合,初步证明所表达的重组蛋白具有良好的体外结合活性;基于制备的能够表达HIV-1 gp120基因的靶细胞模型,运用IFA法开展目的蛋白的细胞结合活性研究,结果显示,目的蛋白能够与靶细胞发生特异性反应,表明成功制备并获得了具有生物活性的新型抗HIV-1蛋白GRFT,为进一步研制新型抗HIV-1基因工程药物及其靶向治疗制剂奠定了坚实基础.     

增强型绿色荧光蛋白的色谱分离和纯化

增强型绿色荧光蛋白(EGFP)是生物领域常用的标记物。在前期成功克隆表达EGFP的基础上,本实验建立了两步分离纯化EGFP的色谱方法,并验证其分离纯化效果,检验EGFP的活性。首先用金属螯合亲和色谱柱HisTrap HP对EGFP的重组菌体破碎上清液进行初步分离,再用葡聚糖凝胶排阻色谱柱Sephadex G-10 HR对其进行脱盐纯化。采用丙烯葡聚糖凝胶排阻色谱柱Sephacryl S-300 HR和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测分离纯化后的EGFP纯度。最后通过荧光分光检测器和非变性聚丙烯酰胺凝胶电泳(Native-PAGE)验证分离纯化后的EGFP是否具有荧光活性。结果表明该方法可以简便快速地分离纯化EGFP,纯度超过98%,同时保持了EGFP的荧光活性。