人TPON端结构域在大肠杆菌中的表达、纯化及活性分析

在利用反转录－ＰＣＲ从人胎肝中获得编码人血小板生成素（ｈＴＰＯ）全长ｃＤ－ＮＡ的基础上,综合ＴＰＯ结构与功能的研究信息,在大肠杆菌中表达了成熟肽Ｎ端结构域．目的蛋白在菌体内以包涵体形式存在,表达量约占菌体总蛋白的３０％;包涵体经变性、复性、凝胶过滤、离子交换层析等步骤处理后,所得产物给Ｂａｂｌ／ｃ小鼠腹腔连续注射８ｄ,第９ｄ摘眼球采血,计数血小板的数量．结果表明,ＴＰＯＮ端结构域具有明显促进血小板生成的作用．     

Gastrointestinal growth factors and hormones have divergent effects on Akt activation

Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigated in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters [CCK, bombesin, carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations (pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations (nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory effects can be seen, which are mediated by different mechanisms.     

Damaging effects of ethylenediaminetetra-acetate and penicillins on permeability barriers in Gram-negative bacteria

1. The permeability barrier against benzylpenicillin has been found to be passive in four strains of penicillinase-producing Gram-negative bacteria (three of Klebsiella aerogenes and one of Escherichia coli). 2. If the three K. aerogenes strains are grown in the presence of sub-inhibitory concentrations of benzylpenicillin, ampicillin or phenethicillin the resultant bacterial cells have deficient permeability barriers. Concentrations of ampicillin or benzylpenicillin less than one-tenth of those required to inhibit growth cause destruction of more than half the permeability barrier in these strains. 3. Benzylpenicillin, ampicillin and phenethicillin have no effect upon the permeability barriers of resting cells from the three K. aerogenes strains. 4. Treatment of resting cells with trisodium EDTA, although failing to sensitize K. aerogenes to lysozyme, severely damages permeability barriers in this species. 5. The magnesium and calcium salts of EDTA do not have the same capacity as the sodium salt for causing damage to permeability barriers in K. aerogenes and E. coli. Damage caused by trisodium EDTA can be at least partially reversed by treatment with Ca(2+) or Mg(2+) ions. It is suggested that EDTA damage is caused by removal of either Ca(2+) or Mg(2+) ions, or both, from the bacterial cell envelope. 6. Bacterial cells with deficient permeability barriers as a result of either growth in the presence of a penicillin or treatment with EDTA remain viable, and revert to their usual permeability after growth in nutrient broth.     

人肿瘤抑素(Tumstatin)在E.coli中的克隆、表达及活性分析

从人胚肾2 93细胞中扩增肿瘤抑素(tumstatin)基因,进行原核表达,纯化和生物活性检测.利用原核表达载体pMAL c2在大肠杆菌BL2 1中表达肿瘤抑素,经AmyloseResin亲和层析柱和QSepharoseFastFlow柱纯化,通过体外内皮细胞增殖、内皮细胞凋亡和鸡尿囊绒膜新生血管生成试验检测其抑制活性.MBP tumstatin在BL2 1中表达率约2 0 % ,肿瘤抑素纯度可达95 % .肿瘤抑素可明显抑制内皮细胞增殖(IC50 约为15 μg ml)、诱导内皮细胞凋亡和抑制鸡尿囊绒膜新生血管生成.研究结果表明,肿瘤抑素对内皮细胞具有明显的抑制作用,提示其在肿瘤治疗中有潜在的应用前景.     

Allosteric and non-allosteric E. coli phosphofructokinases: effects on growth.

In wild type $\text{Escherichia coli}$ K-12 ca. 90% of phosphofructokinase is known to be the allosteric enzyme Pfk-1, and the rest is Pfk-2, a non-allosteric enzyme. An isogenic strain series has now been constructed with varying combinations and amounts of Pfk-1 and Pfk-2 (e.g., no Pfk-1, high level of Pfk-2; normal level Pfk-1, high level Pfk-2, etc.). In minimal medium with glucose, glucose-6-P, and glycerol, aerobically and anaerobically, provided there is adequate total amount of enzyme, what allosteric type it is does not make much difference to growth rate or yield of this organism.     

人骨骼肌肌钙蛋白C在大肠杆菌中的表达及其抗肿瘤作用

为了探索重组人骨骼肌型肌钙蛋白C(rhTnC)的抗肿瘤作用,采用PCR技术,从人乳腺cDNA文库中获得编码TnC的cDNA序列,在大肠杆菌中进行了表达.经镍柱亲和层析纯化后,分别检测其在体外对人脐静脉内皮细胞生长的抑制作用与对小鼠异位移植肿瘤(S-180)生长的抑制作用.结果表明,rhTnC在大肠杆菌中呈可溶性表达.分离纯化后的rhTnC在体外对人脐静脉内皮细胞生长呈剂量依赖性抑制(IC50为7.5μg/ml);对小鼠异位移植肿瘤的生长也有明显的抑制作用,在20mg/(kg·d)剂量下对S-180的抑制率为64.4%.以上结果说明,rhTnC可抑制血管内皮细胞生长,并具有抗肿瘤作用.     

Biological and chemical characteristics of allergen preparations from Neisseria (N. meningitidis, N. gonorrhoeae, N. perflava)]

Various methods of isolating allergen fractions from N. meningitidis, N. gonorrhoeae and N. perflava were tested. The biological activity of the preparation was found to depend on the method of its production, which determined its chemical composition. When gonococcal and meningococcal allergens and N. perflava allergen were used in skin tests, cross reactions were observed. Nevertheless, as the intensity and size of skin reaction was much greater when a homologous preparation was administered, it was possible to differentiate the presence of sensitization to a definite microbial species. Electrophoresis in acrylamide gel revealed the heterogeneity of allergen preparations. The ability of the preparation to induce skin reaction was not connected with its serological properties.     

重组人胰激肽原酶复性工艺的研究

目的:研究重组人胰激肽原酶包涵体变性及复性的工艺。方法:对本实验室构建的重组人胰激肽原酶大肠杆菌进行IPTG诱导表达表达成功后,菌体经超声破碎释放包涵体,包涵体经洗涤、变性、稀释和尿素梯度凝胶过滤色谱这两种方法复性后(Sephadex-G75),通过测定酶活检验复性效果。结果:①重组人胰激肽原酶工程菌经过IPTG诱导后能够表达目的蛋白,目的蛋白以包涵体形式存在,将细胞破碎后,包涵体经过3次洗涤,纯度达到71.93%;②变性包涵体经24小时稀释复性后,蛋白浓度达到72.61μg/m L,酶的比活达到13.84 U/mg;③变性包涵体经过2个小时的尿素梯度凝胶过滤复性后,蛋白浓度可达到830.07μg/mL,酶的比活达到48.61 U/mg。结论:两种复性方法均可以使包涵体达到一定的浓度和比活,比较发现尿素梯度凝胶过滤色谱具有复性时间短和比活力高等优点,可作为重组人胰激肽原酶复性的一种有效的手段。     

Adaptation to sulfonamide resistance in Neisseria meningitidis may have required compensatory changes to retain enzyme function: kinetic analysis of dihydropteroate synthases from N. meningitidis expressed in a knockout mutant of Escherichia coli.

Previously, the effects of three point mutations (at amino acid positions 31, 84, and 194) in the gene coding for a sulfonamide-resistant dihydropteroate synthase of Neisseria meningitidis were analyzed by site-directed mutagenesis. Changes at positions 31 and 194 abolished the phenotypic expression of sulfonamide resistance, while a change at position 84 appeared to be neutral. These studies are here extended to correlate the alterations in phenotype with effects on enzyme kinetics by expressing the cloned meningococcal genes in an Escherichia coli strain that had its dhps gene partially deleted and replaced by a resistance determinant. The most dramatic effects were produced by mutations at position 31. A change from the Leu found in the resistant isolate to a Phe (the residue found in sensitive isolates) led to a 10-fold decrease in the Km and a concomitant drop in the Ki. Changes at position 194 also affected both the Km and Ki but not to the same extent as mutations at position 31. Changing position 84 altered the Km only slightly but significantly. This latter change was interpreted as a compensatory change modulating the function of the enzyme. In another type of resistance gene, 2 amino acid residues, proposed to be an insertion, were deleted, resulting in a sensitive enzyme. However, the resulting Km was raised 10-fold, suggesting that compensatory changes have accumulated in this type of resistance determinant as well. This resistance gene differs by as much as 10% from the sensitive isolates, which makes identification of important mutations difficult.     

Involvement of chlA, E, M, and N loci in Escherichia coli molybdopterin biosynthesis.

All molybdenum enzymes except nitrogenase contain a common molybdenum cofactor, whose organic moiety is a novel pterin called molybdopterin (MPT). To assist in elucidating the biosynthetic pathway of MPT, two MPT-deficient mutants of Escherichia coli K-12 were isolated. They lacked activities of the molybdenum enzymes nitrate reductase and formate dehydrogenase, did not reconstitute apo nitrate reductase from a Neurospora crassa nit-1 strain, and did not yield form A, a derivative of MPT. By P1 mapping, these two mutations mapped to chlA and chlE, loci previously postulated but never definitely shown to be involved in MPT biosynthesis. The two new mutations are in different genetic complementation groups from previously isolated chlA and chlE mutations and have been designated as chlM and chlN (closely linked to chlA and chlE, respectively). The reported presence of Mo cofactor activity in the chlA1 strain is shown to be due to in vitro synthesis of MPT through complementation between a trypsin-sensitive macromolecule from the chlA1 strain and a low-molecular-weight compound from the nit-l strain.     

人己糖胺酶D的原核表达、纯化及酶学特性研究

糖基化修饰是一种重要的蛋白质翻译后修饰,参与生物体中的信号传导、细胞识别等多种细胞活动,糖基缀合物的正常水解是生物体代谢的必需途径.人己糖胺酶D( Hexosaminidase D)是新发现的一种存在于人细胞质中的切除GalNAc糖基化修饰的外切酶,但该酶的酶学特性尚不清楚.利用PCR的方法,将Hex D的cDNA序列构建到质粒pET3C中,重组质粒转化大肠杆菌BL21( DE3) plysS后,通过优化异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度(0.1mmol/L)和诱导时间(10 h)获得了高可溶性表达的重组蛋白酶.采用Ni-NTA亲和层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小(58 kDa)和纯度(95％以上).以4-甲基伞形酮-2-乙酰氨基-2-脱氧半乳糖(4-MU-O-GalNAc)为荧光底物,测定该酶的最适反应pH值为5.5,最适反应温度为37℃,且该酶的热稳定性较好,在50℃下放置半小时仍有较高活性,1mmol/L的金属离子(CuSO4、FeSO4·7H2O、MgCl2· 6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)及EDTA对该酶活性影响不大,10mmol/L AlCl3、CuSO、FeSO4·7H2O对该酶有不同程度的抑制.在最适条件下(pH 5.5,37℃)下,该酶的Km为0.16mmoL/L,最大反应速率为3.06 μmol/( min·mg).     

Isolation of E. coli from foam and effects of fluoroquinolones on E. coli and foam production in male Japanese quail

Sexually active male Japanese quail (Coturnix coutrnix Japonica) produce a foamy substance from their cloacal gland. It was postulated that bacteria played an important role in production of foam. The primary objective of this study was to isolate and identify bacteria present in the cloacal foam. The secondary objective was to evaluate the effect of fluoroquinolone treatment on bacterial counts and foam production. Healthy adult Japanese quail were maintained in individual cages under uniform husbandry conditions and allocated arbitrarily into three groups (each group consisted of 12 male and 12 female birds). Foam was collected from the cloacal gland of male birds of each group separately into sterile petri dishes and was cultured to isolate and identify bacteria and to determine their sensitivity to various antibiotics. Escherichia coli bacteria, sensitive to various antibacterials (including the fluoroquinolones ciprofloxacin and pefloxacin), were isolated. In the second part of the study, male quails of Group I (control) received I mL vehicle (normal saline 0.9% (w/v) NaCl) daily (via the intraperitoneal route) for 12 days. Male birds from groups II and III were treated intraperitoneally with ciprofloxacin or pefloxacin at the rate of 10 mg and 12 mg per/kg body weight respectively, for 12 days. In antibiotic-treated birds, there was a gradual reduction in foam production during treatment. At the end of treatment, the cloacal gland area was smaller (P < 0.05) in pefloxacin-treated birds compared to the other groups. Furthermore, a trend towards decreasing body weight and fertilizing ability was noted in the same group. A drastic reduction in bacterial counts of foam was recorded only in fluoroquinolone-treated groups during treatment period. After cessation of treatment, all end points were increasing back to pre-treatment levels. In conclusion, E. coli were present in the foam of the cloacal gland of Japanese quail and may have a role in foam production.     

E．coli分泌表达载体的构建和人表皮生长因子在E．coli中的分泌性表达

采用ＰＣＲ技术从Ｅ．ｃｏｌｉ基因组片段中克隆出碱性磷酸酯酶（ＰｈｏＡ）的启动子和信号肽序列．在ＰｈｏＡ启动予５＇端设计了ＥｃｏＲⅠ酶切位点,在信号肽编码序列３＇端设计了ＨｉｎｄⅢ酶切位点．将ＰＣＲ产物酶切后ＥｃｏＲⅠ－ＨｉｎｄⅢ片段克隆至ｐＢＲ３２２的ＥｃｏＲⅠ－ＨｉｎｄⅢ位点,组构出含有ＰｈｏＡ启动子和信号肽序列的分泌表达载体ｐＢＭ－Ｐｈｏ－１．之后将人表皮生长因子的成熟肽基因克隆至该载体,使之在Ｅ．ｃｏｌｉ中获得分泌表达,另采用ｐＩＮⅢ载体系统以分泌方式表达了人表皮生长因子。     

霍乱弧菌脂多糖O抗原基因在大肠杆菌中的克隆及表达

经典生物型及埃尔托生物型霍乱弧菌的染色体DNA片段分别与载体质粒pUC18,B.S(M13~+)进行克隆,从克隆株中筛选到能表达霍乱弧菌脂多糖O抗原基因的重组子。它们所表达的脂多糖O抗原具有很好的抗原性及免疫原性,其重组质粒pMG-301、pMG-302经酶切分析表明,外源片段大小分别为8.4kb,7.6kb,较文献报道的16kb要小,而且基因结构之间也存在很大差异。