脂肪酶Novozyme435手性拆分(R,S)-扁桃酸

扁桃酸是一种重要的医药中间体,本文利用固定化脂肪酶Novozyme435作为催化剂,乙酸乙烯酯作为底物,异丙醚作为溶剂,发现当乙酸乙烯酯与扁桃酸摩尔浓度比为6,扁桃酸浓度为180mmol/L,反应温度50℃条件下底物的转化率为50％,eep(产物对应体过剩量)和ees(底物对应体过剩量)均99.9％以上,结果表明脂肪酶Novozyme435对于扁桃酸具有好的选择性.     

人EC-SOD在大肠杆菌中表达的发酵条件研究

对含人EC-SOD重组质粒的工程菌进行诱导表达形成包涵体.通过SDS-PAGE电泳分析,考察了诱导时间和诱导剂种类、浓度对EC-SOD表达量的影响.同时考察了细胞培养温度对产物表达形式的影响,并对菌体密度不同时诱导表达的结果进行了比较.     

重组人血管内皮生长因子(hVEGF121)工程菌的发酵及表达产物的分离纯化

该研究优化了重组人血管内皮生长因子hVEGF121工程菌的发酵条件，考察乳糖诱导浓度和时间对目的蛋白表达量的影响。经乳糖诱导，目的蛋白以包涵体形式存在。通过菌体裂解、包涵体洗涤、裂解等初步纯化后，再经过DEAECellulose离子交换柱纯化和复性，经SDS-PAGE分析显示得到电泳纯的hVEGF121。     

岩黄连药材的提取工艺研究

［摘要］目的正交实验优选岩黄连药材的最佳提取工艺。方法以岩黄连中总生物碱、脱氢卡维丁的含量为指标,比较乙醇和酸水两种不同提取溶媒以及回流、渗漉两种不同提取方法,采用L9（34）正交设计,对影响岩黄连提取工艺中的溶剂浓度、溶剂用量、提取时间、提取次数等4个因素进行优化筛选。结果乙醇提取优于酸水提取,回流法提取效果优于渗漉法。醇提最佳工艺条件为：用75%乙醇提取,每次加醇量10倍,提取3次,每次提取时间2 h。结论优选得到的工艺简便、稳定、可行。     

重组人血管内皮抑素的阳离子交换色谱复性与纯化条件研究

对rhEndostatin包涵体蛋白的色谱复性条件进行研究,摸索出最佳的复性、纯化条件,解决其在大肠杆菌中表达提取时遇到的蛋白变性问题。按实验条件对表达的rhEndostatin包涵体蛋白进行提取、洗涤、变性;利用正交法设计重组蛋白的色谱复性、纯化实验;利用SP-Sepharose Fast Flow阳离子交换柱对rhEndostatin包涵体蛋白进行了色谱复性、纯化。最佳复性条件为rhEndostatin上样浓度为2.5 mg/mL,洗脱体积流量为1/20床体积/min(3.5 mL/min),NaCl浓度0.1 mol/L,GSH/GSSG的浓度比为3/0.3 mmol/L。在此条件下纯度约97%,蛋白回收率为90%,重组蛋白的活性最高,IC50达到5μg/mL。     

岩黄连生物碱提取工艺研究

目的：正交优选岩黄连的最佳提取工艺。方法：以岩黄连中总生物碱、脱氢卡维丁的含量为指标,比较乙醇和酸水两种不同提取溶媒以及回流、渗漉两种不同提取方法并采用L9（3^4）正交设计,对影响岩黄连提取工艺中的溶剂浓度、溶剂用量、提取时间、提取次数四个因素进行优化筛选。结果：乙醇提取优于酸水提取,回流法提取效果优于渗漉法。醇提最佳工艺条件为A2B2C3D2,即75％乙醇提取,每次加醇量10倍,提取3次,每次提取时间2h。结论：优选得到的工艺简便、稳定可行。     

重组人血管生长素包涵体复性条件研究

目的摸索重组人血管生长素(rhANG)复性影响条件,以提高rhANG的复性效率。方法超声波破碎菌体,变性萃取rhANG包涵体。分析rhANG包涵体起始浓度、氧化型、还原型谷胱甘肽比例、盐酸胍和添加精氨酸对复性的影响。结果在0.1 mol/L Tris-HCl pH 7~8,还原型、氧化型谷胱甘肽比率为5,盐酸胍为0.2~0.5 mol/L,起始蛋白质浓度0.1~0.2 mg/mL时,rhANG蛋白复性率为60%;0.5 mol/LL-精氨酸有助于复性。结论初步优化rhANG包涵体复性参数,为rhANG的放大制备创造了条件。     

盐酸丁卡因注射液在气管内插管气囊中的释放

目的:研究气管内插管气囊中的盐酸丁卡因在体外条件下的释放行为。方法:采用高效液相色谱法测定盐酸丁卡因的含量,分别采用静态累积释放和交替洗脱释放的方法观察盐酸丁卡因的释放过程,并分析了pH、溶剂极性对2%盐酸丁卡因释放的影响;比较了不同浓度盐酸丁卡因对累积释放量和洗脱释放量的影响。结果:2%盐酸丁卡因4.5h后在85mL溶剂质量浓度为(5.76±0.19)mg.L-1(n=5),4.5h时仍稳定释放;pH、溶剂极性对盐酸丁卡因的释放影响显著(P<0.01)。浓度较高盐酸丁卡因有较快的释放速率,有较大的累积释放量和洗脱释放量。结论:盐酸丁卡因在气囊中能稳定释放,释放过程受pH、溶剂极性及盐酸丁卡因浓度的影响。     

蚕沙叶绿素提取及合成脱镁叶绿酸工艺改进研究

目的探讨不同提取工艺对蚕沙中提取叶绿素及制取叶绿素衍生物脱镁叶绿酸的影响,以提高蚕沙叶绿素及叶绿素衍生物的制备量。方法单因素实验设计,以蚕沙软化时间(含水量)、不同提取溶剂为考察条件,优化蚕沙叶绿素提取工艺,并探讨浓盐酸脱镁时间对叶绿素合成脱镁叶绿酸制备率的影响。结果蚕沙软化时间2 h(含水量26%)、丙酮∶乙醇体积比1∶1做溶剂为提取叶绿素的最佳条件,提取率提高到1.43%。以蚕沙叶绿素粗品合成叶绿素衍生物脱镁叶绿酸,浓盐酸脱镁搅拌反应72 h制备率达670 mg.g 1。结论通过改良工艺,提高了蚕沙叶绿素及其衍生物脱镁叶绿酸的提取制备率。     

人胰岛素原在基因工程菌中的高效表达

目的　构建RRhPI-pQE4. 0E .coliM15表达系统 ,以高效表达 (His) 6 -Arg -Arg -人胰岛素原。方法　正交法优化发酵条件 ,通过湿菌体收率、包涵体收率及发酵液A60. 0 的测定对优化结果进行评估 ,并进一步分析发酵过程中主要因素对高密度发酵和高效表达的影响。结果　发酵条件优化后 ,每升发酵培养基可得湿菌体约 4 3g ,包涵体约 14g(湿重 )。结论　优化发酵条件可使湿菌体及包涵体的收率提高。     

抗还原并烷化的AChE单克隆抗体识别的抗原决定基(英文)

电器官还原并烷化的乙酰胆碱酯酶(RA-A ChE)的溴化氰裂解肽或又经胃蛋白酶切割的肽与抗RA-AChE单克隆抗体E9、F6及F12仍有抗原抗体反应。经溴化氰及胰蛋白酶割的肽与3者反应消失。氧化破坏RA-AChE糖侧链、溴化氰裂解的RA-A ChE又经酶切除糖侧链后,与3者反应无变化。E9、F6及F12所识别的抗原决定簇是多肽型,不是多糖型。     

The [173-196] fragment of ovalbumin suppresses ovalbumin-specific rat IgE responses

Peptides and protein hydrolysates are attractive tools for the induction of tolerance or regulation of targeted B and/or T cell responses. In vivo, peptides are mainly produced by the action of digestive enzymes or following the processing of exogenous antigens by antigen-presenting cells (APCs). In vitro, these molecules are generally produced by enzymatic digestion and chemical hydrolysis of proteins. We investigated the T and B cell determinants of the major food allergen ovalbumin (nOVA) in rat by analyzing (1) the stimulatory effect of nOVA peptides generated by cyanogen bromide (CNBr) cleavage on nOVA-specific T cells, and (2) the potential of CNBr-derived OVA fractions to induce oral tolerance to nOVA. Peptide fractions of the CNBr-hydrolysated OVA were isolated by high-pressure liquid chromatography and tested for their ability to stimulate nOVA-specific T cells isolated from rats parenterally immunized with nOVA. The nOVA fractions containing the stimulatory determinants were then intragastrically administered to rat to test their potential to induce oral tolerance. The hole CNBr hydolysate stimulated proliferation of nOVA-specific T cells. Three out of the five HPLC-purified peptidic fractions were also able to stimulate proliferation and cytokine production by nOVA-specific T cells. A peptide fraction exhibiting a single peak by HPLC contained the 173-196 nOVA segment and stimulated nOVA-specific T cells. This segment also promoted oral tolerance to nOVA and reduced IgE responses. CNBr hydrolysis releases several peptides with stimulatory effect on nOVA-specific T cells including a new nOVA [173-196] T cell determinant which induces oral tolerance to nOVA.     

C群脑膜炎球菌结合疫苗的制备及其免疫原性

 目的  制备由不同分子大小C群脑膜炎球菌多糖（group C meningococcal polysaccharide,CPS）与破伤风类毒素（tetanus toxoid,TT）共价偶联而成的结合疫苗（CPS-TT）,并检测其在小鼠中的免疫原性。 方法   将CPS降解成不同大小的片段,经碳二亚胺（carbodiimide,EDAC）或溴化氰（cyanogen bromide,CNBr）活化后偶联到TT上制成CPS-TT。NIH小鼠皮下注射制备的不同CPS-TT,用ELISA法测定小鼠血清抗CPS和TT IgG抗体。 结果   EDAC活化的降解CPS的衍化率（3.9%～5.7% ）和多糖回收率（11.4%～21.3%）均高于CNBr活化的降解CPS的衍化率（0.4%～1.2%）和回收率（2.4%～11.5%）。EDAC或CNBr活化的降解CPS制备的CPS-TT在小鼠中均具有较强的免疫原性,但EDAC活化的降解CPS制备的CPS-TT免疫小鼠的抗CPS抗体滴度高于CNBr活化的降解CPS制备的CPS-TT免疫小鼠。结论  EDAC活化的降解CPS可用于制备C群脑膜炎球菌结合疫苗。     

Total synthesis of horse heart cytochrome c

A peptide corresponding to the native (1–66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met⁶⁵-Glu⁶⁶ peptide bond with simultaneous transformation of the Met⁶⁵ residue into the activated C-terminal [Hse⁶⁵lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66–104) to produce fully synthetic [Hse⁶⁵]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.     

TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca2+-activated K+ channels.

The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.     

Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium

Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (71-70 fragment) and SV-IV/B (71–90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites. In contrast, only SV-IV/A was shown to possess, like SV-IV, the property of inhibiting both the intensity of the carrageenin-induced rat foot edema and the platelet aggregation induced in vivo by different agents. Finally, the two protein fragments were found to be completely unable to inhibit both the mitogen-induced proliferation of human T cells and the mixed lymphocyte reaction.     

Influence of minor groove binders on the eukaryotic topoisomerase II cleavage reaction with 41 base pair model oligonucleotides

Summary This report deals with the cleavage reaction of calf thymus (CT) topoisomerase II with oligonucleotides containing one main cleavage site and adjacent binding sites for minor groove binders. The sequences of the oligonucleotides were derived from a pBR 322 sequence, which contains one main topoisomerase II cleavage site. The cleavage reaction was performed under increasing concentrations of minor groove binders and it showed characteristic inhibition dependences of topoisomerase II to the binding sites and to the binding length of the minor groove binders. The extension of the minor groove binder length on DNA from 4 to 10 base pairs (bp) by netropsin and bis-netropsin, respectively, causes a strong increase of the topoisomerase II cleavage inhibition. The same is observed by the introduction of a second minor groove binder sequence symmetrically positioned around the topoisomerase II main cleavage site. The combination of two different minor groove binders can lead to an increased topoisomerase II inhibition but also to a prevention of total inhibition as shown with chromomycin A₃ and distamycin A at concentrations of 0.1 and 0.25 M, respectively.     

Isolation and characterization of a neurophysin from ostrich neurohypophyses

A neurophysin has been isolated from ostrich neurohypophyses using acid acetone extraction, salt fractionation and Sephadex G-75 chromatography. The crude neurophysin eluting from the Sephadex G-75 column was subjected to a) reverse-phase HPLC followed by Sephadex G-75 chromatography, b) DEAE-Sephadex A-50 chromatography or c) isoelectric focusing. The different homogeneous ostrich neurophysin fractions so obtained were compared i.t.o. amino acid composition, spectral properties, N-terminal amino acid residues and PAGE. They all revealed a single N-terminal Ala residue and displayed spectral properties (A₂₈₀/A₂₆₀ < 1) which are typical of mammalian neurophysin-like polypeptides. Ultracentrifugation studies on purified ostrich neurophysin over a range of concentrations revealed a reversible concentration dependent association behaviour characterized by the presence of dimeric complexes at higher concentrations. Partial sequencing from the N-terminus revealed the molecule to be VLDV-like. The purified molecule was also submitted to CNBr fragmentation.     

Photoreactive derivative of Kunitz's soybean trypsin inhibitor.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1–114 and 115–181.Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [³H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.