重组鲈鱼生长激素的酶联免疫测定方法

建立了重组鲈鱼生长激素（jspGH0的间接ELISA，以jspGH的小鼠多克隆抗体作为一抗，HRP羊抗鼠IgG作为二抗，在抗体及酶标二抗的稀释液中加入4％PEG，从而提高检测灵敏度至40ng/ml，抗原浓度在40－1000ng/ml范围内与OD450nm值成线性关系。此法较简便，灵敏，可用于检测基因工程菌发酵液中的jspGH含量。     

酵母表达的重组草鱼生长激素对鲤鱼的促生长效应

通过发酵罐放大培养,优化发酵参数,提高了重组草鱼生长激素在酵母中的表达量。以含重组草鱼生长激素的酵母裂解物投喂鱼苗,实验组鱼体重的增加显著高于对照组,体长的亦高于对照组。鱼体生化组成分析表明：投喂转草鱼生长激素基因酵母裂解物能显著提高鱼体的干物质和蛋白含量,并降低鱼体脂肪酸含量。     

重组植物毒素gelonin的纯化及性质研究

采用pET28a载体系统在E.coli BL21中进行了植物毒素gelonin的原核表达，产物经SP-Sepharose，Superdex 75二步纯化，获得了电泳纯的重组植物毒素gelonin。将重组的gelonin与从植物种子中提取的天然gelonin进行了Western blot，ELISA，无细胞体系中蛋白质合成抑制实验以及超螺旋DNA裂解研究。结果都表明，重组植物毒素gelonin与天然gelonin具有相似的生物活性。     

冷藏过程中调味料对海鲈鱼片品质的影响

目的 研究复合调味料对海鲈鱼片贮藏品质的影响,以期为海鲈鱼的保鲜提供理论指导和技术支持.方法 以海鲈鱼片为研究对象,采用花椒、桂皮、八角、白酒、生姜、食盐等6种调味料及其复合物的溶液浸渍海鲈鱼片,测定4℃贮藏条件下海鲈鱼片的鲜度和质构指标的变化.结果 单一调味料液处理可使鱼片的鲜度和质构指标变化减缓,其中桂皮调味料液处理有效延缓了鱼片的菌落总数、pH值、硫代巴比妥酸(TBA)值、挥发性盐基氮(TVB-N)值和K值的上升.经复合调味料液处理后,鱼片的各项鲜度指标均有所改善,且自由水的含量显著降低,持水力比未处理鱼片高出8％,改善了鱼肉的持水性.结论 6种调味料复合后具有协同抗菌性能,保鲜性能较好,可有效延缓海鲈鱼的蛋白质分解和脂肪氧化,延长货架期至12 d.     

吸血蝙蝠纤溶酶原激活剂α2的原核表达及抗体制备

利用人工合成的吸血蝙蝠唾液纤溶酶原激活剂α2(DSPAα2)基因,研究了其在细菌中的表达及表达产物的纯化和抗体制备.首先人工合成DSPAα2基因,并构建原核表达载体转化大肠杆菌.经IPTG诱导后,DSPAα2在细菌中得到了高效表达.SDS-PAGE结果显示,以包涵体的形式存在的DSPAα2重组蛋白占到细菌总蛋白的32%.包涵体裂解后经亲合层析柱纯化,100mL菌液中能得到2.2mg纯的重组蛋白.用纯化的DSPAα2分别免疫大鼠和小鼠,经ELISA检测,获得了效价达到1∶12800以上的高质量的抗血清.Western blot结果显示抗体能与DSPAα2特异性地结合.     

中国明对虾蜕皮抑制激素在大肠杆菌中的表达与分离纯化

采用大肠杆菌表达系统对中国明对虾蜕皮抑制激素(MIH)进行了体外重组表达研究.重组蛋白以包涵体的形式存在于菌体中.尿素-SDS-PAGE分析表明,经1mmol/L的IPTG诱导4h后,重组蛋白获得了大量表达,在分子量为13 kD处有一条与预测大小一致的特异性蛋白条带;经金属螯合柱纯化后,得到了电泳纯的融合蛋白.Western blotting检测结果表明:重组表达的融合蛋白与兔抗刀额新对虾MIH的多克隆抗体特异结合,证实该融合蛋白为中国明对虾MIH.通过胶内酶切与LC-ESI-MS分析进一步证实融合蛋白的部分肽段与日本对虾MIH一致.MIH融合蛋白的成功表达为进一步深入研究其在中国明对虾蜕皮过程分子作用机制奠定了基础.     

八肋游仆虫一种富含谷氨酸的蛋白GARP的表达与纯化

为研究游仆虫中含有三核苷酸重复序列的GARP基因编码的GARP蛋白在细胞中的功能,利用定点突变技术,将GARP基因中的TGA突变为通用半胱氨酸密码子TGT.突变后的GARP基因构建于原核表达载体pRSETc中,得到重组质粒pRSETc-GARP,将pRSETc-GARP质粒转化至大肠杆菌BL21(DE3)中,经IPTG诱导,带有纯化标签的重组蛋白His6-GARP获得可溶表达,表达产物与抗His抗体在约26 kDa处有很强的交叉反应.His6-GARP蛋白在不同pH条件下通过两次阴离子交换层析和一次凝胶层析三步纯化达到电泳纯.     

N—氨甲酰基—D—氨基酸酸胺水解酶基因的克隆与表达

按本室纯化的N-氨甲酰基-D-氨基酸酰胺水解酶（DCase)的N-末端氨基酸序列设计了正向引物，并在巳报道的不同来源菌株DCase的氨基酸序列同源性分析的基础上，设计了反向引物。用菌株No.2262总DNA为模板，经PCR扩增获得长度为约0.9kpb的片段，DNA序列分析表明基因全长为912bp。将该片插入PET-3a，构建成重组子pET-DCase，并在E.coliDH5a中获得表达，经SDS-PAGE检测，表达产物分子量与天然纯DCase相同，约为35kd.     

热诱导FLP重组酶删除转基因烟草外源基因

利用热激启动子Hsp18.2、FLP重组酶基因及其专一识别位点构建了重组酶删除系统的植物表达载体转化烟草.热激处理后转基因植株中FLP重组酶表达并识别两个同向loxP-frt重组酶识别位点,使两位点间的DNA插入片段(包含kn1、gus、nptⅡ基因)从转基因烟草基因组中删除,由kn1基因引起的转基因烟草特殊表型及gus基因产生的蓝色表型消失.     

N-氨甲酰基-D-氨基酸酰胺水解酶基因的克隆与表达

按本室纯化的N 氨甲酰基 D 氨基酸酰胺水解酶 (DCase)的N 末端氨基酸序列设计了正向引物 ,并在已报道的不同来源菌株DCase的氨基酸序列同源性分析的基础上 ,设计了反向引物。用菌株No . 2 2 62总DNA为模板 ,经PCR扩增获得长度为约0 9kpb的片段 ,DNA序列分析表明基因全长为 912bp。将该片段插入 pET 3a ,构建成重组子 pET DCase ,并在E coliDH5α中获得表达 ,经SDS PAGE检测 ,表达产物分子量与天然纯DCase相同 ,约为 35kD。     

利用自剪切融合蛋白在大肠杆菌中表达并纯化兔防御素

将源自兔嗜中性细胞的防御素基因NP-1插入原核表达载体pTYB2,并将重组质粒pTYB2-NP1转入大肠杆菌BL21中,用IPTG诱导使防御素以融合蛋白的形式表达。然后,通过亲和层析利用自剪切融合蛋白分离提纯目的蛋白。蛋白电泳检测到以二聚体形式存在的防御素,但此蛋白没有对大肠杆菌的抑菌活性。同时,对该表达纯化系统的特点和用原核生物大肠杆菌表达真核生物防御素的问题进行了分析和讨论。     

Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry, and chemiluminescence detection

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.     

海洋弧菌几丁质酶的产酶条件及分离纯化研究

将产几丁质酶的海洋弧菌CY01菌株进行发酵培养，对产酶的培养条件等进行了优化。发酵液经硫酸铵分级盐析，几丁质亲和层析，Sephadex G-100、Sephacryl S-200凝胶分离纯化，分离出三种几丁质酶Chi1，Chi2，Chi3，采用SDS－PAGE测得三种酶的分子量分别为27kD，39kD和46kD。     

The recombinant human dentin matrix protein 1-coated titanium and its effect on the attachment, proliferation and ALP activity of MG63 cells

The aim of the present work was to design a bio-interactive implant surface by coating recombinant human dentin matrix protein 1 (hDMP1) onto titanium and to investigate the biological function of this material. Firstly, the plasmid containing the hDMP1 cDNA was constructed and hDMP1 was expressed, purified and characterized. Then, hDMP1 was coated onto the surface of Ti substrates via a biochemical technique and the procedure was divided into three steps: in the beginning, titanium was treated by regular polishing and denoted as Cp-Ti; then, Cp-Ti received alkaline and water treatment and was nominated as AW-Ti; finally, AW-Ti was coated with hDMP1 and referred to as hDMP1-Ti. The inserts of hDMP1 genes were detected by enzyme digestion as well as gel electrophoresis, and the complete nucleotide sequence of hDMP1 was tested. The purified recombinant hDMP1 was electrophoresed on a 10?% SDS-PAGE gel. Cp-Ti, AW-Ti and hDMP1-Ti were characterized by X-ray photoelectron spectroscope and water contact angles tests. The biological activity of MG63 cells cultured in the three groups was investigated by the cell attachment, proliferation and alkaline phosphatase activity assays. The results show that hDMP1 was successfully constructed and coated onto the titanium surface, and hDMP1-Ti had higher hydrophilicity than Cp-Ti. Compared with Cp-Ti and AW-Ti, hDMP1-Ti showed better in vitro bioactivity.     

Separation and characterization of an IgG2 antibody containing a cyclic imide in CDR1 of light chain by hydrophobic interaction chromatography and mass spectrometry

Hydrophobic interaction chromatography (HIC) was used to separate populations of recombinant IgG2 antibody that were created as a result of prolonged incubation at 40 degrees C. Antibody was separated by HIC into three major and seven minor fractions. All but one fraction was composed of antibody with distinct chemical modifications that resulted from exposure to elevated temperature. The results of intact and reduced mass analysis as well as peptide map data derived from the three major HIC fractions indicated that the antibody was being chromatographically separated into populations containing a succinimidyl intermediate in complementarity determining region 1 (CDR1) on zero, one, and two light chain arms. Lower level species purified by HIC were analyzed by intact and reduced mass analysis and laser-induced fluorescence capillary electrophoresis (CE-LIF) and consisted of an antibody that was clipped in four different places in the heavy chain as well as misfolded and aggregated antibody. The potency of the recombinant antibody containing a succinimidyl intermediate on zero, one, and two light chain arms was analyzed by LANCE binding assay and a cell based in vitro bioassay, and the occurrence of this modification on one or both light chain arms was associated with a reduction in the binding affinity of the molecule to the target by approximately 10%. We show that HIC has the unique ability as a first step purification method to separate populations of antibody which are covalently modified under stability programs. The method conditions that have been developed for the HIC assay are ideal for purifying antibodies with labile modifications for the purpose of further characterization.     

HIV-1中国流行株gp120基因在痘苗病毒中的表达及其与核酸疫苗的实验免疫研究

将HIV－1中国流行株gp120基因在痘苗病毒中进行表达,以期获得重组痘苗病毒,与核酸疫苗混合免疫,评价免疫效果,为艾滋病疫苗开发研制打下基础。将HIV－1中国流行株gp120基因片段插入到pJ38载体启动子下游,经同源重组和血凝素阴性空斑筛选重组痘苗病毒,SDS－PAGE、Western blot检测目的蛋白。以重组病毒和核酸疫苗免疫BALB／c小鼠,进行淋巴细胞转化实验、CTL、CD4＋CD8＋T细胞数目以及血清抗体等细胞免疫和体液免疫指标检测。结果获得的重组痘苗病毒vJ38pg120能够表达Gp120蛋白并诱导机体产生细胞免疫和体液免疫,具有良好的免疫原性,其免疫效果以2rVV-DNA混合方式为最好。重组痘苗病毒vJ38gp120。     

牙鲆生长激素基因的克隆及其在大肠杆菌中的融合表达

采用逆转录聚合酶链式反应(RT-PCR)方法,从牙鲆(Paralichthys olivaceus)脑垂体总RNA中扩增出编码牙鲆生长素(GH)成熟肽基因序列。重组至融合表达载体pGEX-4T-3中,构建成牙鲆GH基因融合表达载体pGEX-gh,转化大肠杆菌BL21(DF3),筛选阳性克隆,IVIG诱导表达。经SDS-PAGE电泳显示在45kD处有特异的蛋白条带出现,该蛋白的表达量随诱导时间的延长而增加,3h达最高值,达到细胞总蛋白的18．3％。该融合蛋白在胞内主要以包涵体状态存在,经优化表达条件,成功地获得了可溶性的融合蛋白,经Glutathione Sepharase 4B凝胶纯化后用Western-blotting检测表明其为牙鲆生长激素,并通过酶联免疫吸附受体法证实其具有生物学活性。     

A TEM study of functionalized magnetic nanoparticles targeting breast cancer cells

In this paper, a transmission electron microscopy (TEM) study was carried out to investigate the ability of magnetic nanoparticles (MNPs) to target breast cancer cells in mice. MNPs were functionalized using Luteinizing Hormone Releasing Hormone (LHRH), whose receptors are expressed in most types of breast cancer cells. LHRH conjugated MNPs (LHRH-MNPs) were injected intravenously into female nude mice bearing MDA-MB-435S.luc tumors for thirty days. These mice were sacrificed 20 h after MNP injection. Tumors and periphery organs including livers, lungs and kidneys were collected for analysis. A dedicated transmission electron microscopy (TEM) study was then carried out to investigate the distribution of nanoparticles in cells. We found that dispersive LHRH-MNPs were distributed in tumor cells and cells in lungs and livers. No LHRH-MNPs were observed in kidney cells. Furthermore, LHRH-MNPs tend to aggregate and form clusters in tumor cells and cells in lungs where metastases were developed. These suggest that MNPs functionalized using LHRH can be used to target both primary cancer cells and the metastatic cells. The study also indicates that TEM is a useful tool to study the sub-cellular distribution of functionalized magnetic nanoparticles in mice bearing breast cancers.     

Rasch measurement in the Assessment of Growth Hormone Deficiency in adult patients

The Assessment of Growth Hormone Deficiency in Adults (AGHDA) questionnaire was previously designed, translated and validated in several European countries to evaluate the impact of the disease on Quality of Life. This study aimed to test the metric properties of the Spanish version by means of Rasch analysis. A sample of 356 consecutive adult patients with untreated GHD was included in the study. Patients responded to the questionnaire at baseline and 12 months apart. Answers were analyzed following the dichotomous logistic response model. Parameter estimates, model-data fit and separation statistics were computed. The invariance of the item parameters across time was tested in the follow-up. Rasch results were additionally employed to ascertain score changes through the calculation of the Reliable Change Index (RCI). Items varied in severity from 8.3 -16.8 units (SE= 0.4-0.5) and fit to define a unidimensional variable. The item separation index (SI)(5.2) indicates a good and reliable (0.9) separation of items along the variable that they define. Moreover, results showed the AGHDA conforms to the model expectation of item parameter invariance between administrations. The substantial (2.3) and reliable (0.8) person SI also suggests the sample was well targeted by the questionnaire. According to the RCI, 84% of the patients did not show a significant transition in their measures. Results denote the items of the AGHDA succeeded in defining a scale characterized by the interval-level of its measures, suggesting the questionnaire could be a useful complement of the clinical evaluation of GHD patients at both group and individual level.