High-level expression of milk-derived antihypertensive peptide in Escherichia coli and its bioactivity

An optimal antihypertensive peptide (AHP), KVLPVP, was linked to form a six-copy of tandem dotetracontapeptide with the specific cleavage site (Arg-X) of clostripain. The gene of the dotetracontapeptide was synthesized and expressed in Escherichia coli BL21. After a 5 h induction with 1.2 mM isopropyl-beta-D-thiogalactopyranoside the recombinant AHP fused with glutathione-S-transferase tag reached the maximal production, 24.6% of total intracellular protein. Following digestion with clostripain and carboxypeptidase B, the product was separated with ultrafiltration and reversed-phase HPLC, and 170 mg of pure recombinant AHP was obtained from 1 L of E. coli culture. The IC50 of the recombinant AHP was 4.6 microM. The systolic blood pressure of spontaneously hypertensive rats could be decreased dramatically by the recombinant AHP in a dose-dependent manner after delivering 0.3 mg of AHP/kg of body weight (BW) or 0.6 mg of AHP/kg of BW orally. The strong antihypertensive effect was reached 4-24 h after oral administration of 0.3 mg of AHP/kg of BW, and the peak point was at the fourth hour (-21.4 +/- 7.2 mm of Hg). This study overcame traditional enzymatic digestion problems in preparing AHP and established a novel approach for industrial production of AHP.     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.     

Wheat bran oil and its fractions inhibit human colon cancer cell growth and intestinal tumorigenesis in Apc(min/+) mice

This study was designed to investigate the cancer preventive activities of wheat bran (WB) oil. We studied the colon cancer preventive effects of WB oil and its subfractions in the Apc(min/+) mouse model, a recognized mouse model for human colorectal cancer, and used human colon cancer cell lines (HCT-116 and HT-29) to identify possible active fractions in WB oil. Our results showed that the oil fraction of WB was more active than the water fraction against the growth of human colon cancer cell lines and that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7% (p < 0.0001) in the Apc(min/+) mouse model. The WB oil was further fractioned into nonpolar lipids and phytochemicals and the phytochemical fraction was fractionated into phytosterols and phytosterol ferulates, 5-alk(en)ylresorcinols, and unidentified constituents by normal phase silica gel column chromatography. Results on cell culture showed that the phytochemical fraction had a higher inhibitory effect on HCT-116 human colon cancer cells than that of WB oil, whereas the nonpolar lipid fraction had less growth inhibitory effectiveness. However, neither fractions showed a stronger inhibition than WB oil in the Apc(min/+) mouse model. The current results demonstrate, for the first time, the intestinal cancer preventive activity of WB oil. The active ingredients, however, remain to be identified.     

Fate of Escherichia coli and Escherichia coli O157 in soils and drainage water following cattle slurry application at 3 sites in southern Scotland

Abstract. Slurry from farm animals may contaminate water supplies, rivers and bathing waters with faecal coliforms, such as Escherichia coli . Where animals harbour the O157 strain the hazard to human health is particularly high, but both the hazard level, and the low incidence and sporadic nature of the excretion of E. coli O157 make it difficult to study this strain under field conditions. The survival of total E. coli and of E. coli O157 were compared in the laboratory for two soils under controlled temperature and moisture. E. coli O157 die-off rate was the same as or quicker than for total E. coli . This result meant that field experiments studying the fate of total E. coli should give a satisfactory evaluation of the risk of water contamination by the O157 strain. In four field experiments at three sites, slurry containing total E. coli numbers of 2.2 × 10⁴ to 5.7 × 10⁵ colony forming units per mL (c.f.u. mL^–1) was applied to drained field plots. Field die-off was faster than expected from laboratory experiments, especially in one experiment where two weeks dry weather followed application. In all but this experiment, the first drain flow events after slurry application led to very high E. coli concentrations in the drains (10³ to 10⁴ c.f.u. mL^–1). E. coli O157 was present in the slurry used for two of the experiments (33 c.f.u. per 100 mL in each case). However the proportion of E.coli O157 was very low (about 1 in 10⁵) and it was not detected in the drainage water. After the first week E. coli drainage water numbers decreased rapidly but they were 1–10 c.f.u. mL^–1 for much of the sampling period after slurry application (1–3 months).     

pfl-act基因在大肠杆菌中的高效表达及其纯化

丙酮酸甲酸裂解酶是肠道细菌在厌氧代谢中十分关键的酶,丙酮酸甲酸裂解酶激活因子(pyruvateform ate lyase activator,PFL-A)在功能上具有重要的作用。为进一步研究PFL-A的激活机理,以大肠杆菌K-12的基因组为模板,通过G enB ank上公布的序列设计引物,扩增出目的基因,克隆到pM D 18-T载体,经测序,所扩增出的基因与p f l-act基因具有99%的同源性。将p f l-act连接到高效表达载体pET-22b( )中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,结果发现,p f l-act以包涵体形式表达。改变诱导剂、诱导剂量或培养温度,对包涵体的形成均没有明显的影响。     

地衣芽孢杆菌GXN151的纤维素酶基因cel12A的序列分析及其在大肠杆菌中的表达

地衣芽孢杆菌菌株GXNl51的cell2A基因为783bp,可编码含261个氨基酸的羧甲基纤维素酶Cel12A,Cel12A的N-末端第1～28氨基酸具典型的信号肽特征、第9～31氨基酸具跨膜功能域特征、第104～261氨基酸形成家族12糖基水解酶(glycosyl hydrolase)功能域。将编码其第73-261氨基酸的DNA序列克隆到大肠杆菌表达载体pET一30a( )得表达质粒pGXNl2A,用1mmol／IPTG诱导处理JMl09(DE3)／pGXNl2A的培养液6h pGXNl2A的表达量达到最高,在JMl09(DE3)和BL21(DE3)pLysS中该表达蛋白质可分别占菌体胞内总蛋白的54．3％和20．9％。在含羧甲基纤维素的LA平板上JMl09(DE3)／pGXNl2A和BL21(DE3)pLysS／pGXNl2A表现有较弱的羧甲基纤维素酶活性,说明重组的Cell2A的催化功能域具有独立的催化活性。包含体检测表明pGXNl2A在JMl09(DE3)中的表达产物大部分形成不溶性包含体。     

Quercetin and its in vivo metabolites inhibit neutrophil-mediated low-density lipoprotein oxidation

This study examined the effects of metabolic transformation of the common dietary flavonoid, quercetin, on its ability to protect low-density lipoprotein (LDL) from neutrophil-mediated modification. Quercetin was shown to be effective in protecting LDL against neutrophil-mediated modification at physiological concentrations (1 microM) and appears to act by inhibiting myeloperoxidase (MPO)-catalyzed oxidation (IC(50) = 1.0 microM). Quercetin was also shown to protect against radical-induced [2,2'-azobis(2-methylpropionamidine)dihydrochloride] oxidation (IC(50) = 1.5 microM). Studies of structure-activity relationships showed that methylation at the 3'-position or glucuronidation at the 3-position did not significantly affect inhibition by quercetin of the MPO activity, but conjugations at both positions significantly reduce its activity. Our results suggest that the common dietary flavonoid, quercetin, and some of its major in vivo metabolites are potential inhibitors of MPO at physiological concentrations. Dietary flavonoids that could modify MPO activity could protect lipoproteins from damage in chronic inflammatory states such as cardiovascular disease.     

甘蓝型油菜中黑芥子酶基因在大肠杆菌中重组质粒的构建和表达

实验通过RT-PCR程序从提取的油菜总RNA中扩增出硫代葡萄糖苷水解酶（又称黑芥子酶,EC3.2.1.147）基因,酶解后插入大肠杆菌表达载体（Escherichia coli）pGEX-4T-1,获得克隆菌株。基因测序在 GenBank中的登陆号为EF583560,翻译的氨基酸序列与已报道的黑芥子酶（GenBank中的登陆号为Q00326）中的一段有一个氨基酸不同,同源性达到99%。经过IPTG诱导表达,在91KDa左右处有表达量很高的一条带。     

人乙醛脱氢酶2的原核表达及其条件优化

为实现人乙醛脱氢酶2（ALDH2）基因在原核生物中高效表达,将含有6×His标签和SUMO融合蛋白标签的人乙醛脱氢酶2基因的表达载体转化至宿主菌BL21（DE3）中。在异丙基硫代-β-D-半乳糖苷（IPTG）诱导下,目的基因在大肠杆菌内高效表达。通过对表达条件的优化,37℃使用终浓度0.3mmol/L的IPTG诱导3h,重组大肠杆菌的表达量可占全菌蛋白的16%。SUMO融合蛋白标签的加入以及较低的诱导温度（16℃）有利于提高人乙醛脱氢酶2基因在大肠杆菌内的可溶性表达。     

Fate of chlorate present in cattle wastes and its impact on Salmonella Typhimurium and Escherichia coli O157:H7

Chlorate salts are being developed as a feed additive to reduce the numbers of pathogens in feedlot cattle. A series of studies was conducted to determine whether chlorate, at concentrations expected to be excreted in urine of dosed cattle, would also reduce the populations of pathogens in cattle wastes (a mixture of urine and feces) and to determine the fate of chlorate in cattle wastes. Chlorate salts present in a urine-manure-soil mixture at 0, 17, 33, and 67 ppm had no significant effect on the rates of Escherichia coli O157:H7 or Salmonella Typhimurium inactivation from batch cultures. Chlorate was rapidly degraded when incubated at 20 and 30 degrees C with half-lives of 0.1 to 4 days. Chlorate degradation in batch cultures was slowest at 5 degrees C with half-lives of 2.9 to 30 days. The half-life of 100 ppm chlorate in an artificial lagoon system charged with slurry from a feedlot lagoon was 88 h. From an environmental standpoint, chlorate use in feedlot cattle would likely have minimal impacts because any chlorate that escaped degradation on the feedlot floor would be degraded in lagoon systems. Collectively, these results suggest that chlorate administered to cattle and excreted in wastes would have no significant secondary effects on pathogens present in mixed wastes on pen floors. Lack of chlorate efficacy was likely due to low chlorate concentrations in mixed wastes relative to chlorate levels shown to be active in live animals, and the rapid degradation of chlorate to chloride at temperatures of 20 degrees C and above.     

Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation

A cDNA encoding mature human tyrosinase was cloned into pET-23a(+) and transformed into E. coli BL21(DE3). Three major recombinant proteins, mature human tyrosinase (RHT??????), N-terminal truncated human tyrosinase (RHT???????), and β-lactamase, were overexpressed as inclusion bodies in E. coli after 12 h of induction with 1.0 mM isopropyl-β-D-thiogalactopyranoside at 37 °C. After sonication and centrifugation, the inclusion body was harvested, solubilized, dialyzed, and refolded into the active form with monophenolase and diphenolase activities. It was purified to homogeneity by DEAE-Sepharose FF and Sephadex G-75. The molecular mass and N-terminal sequence were 57.0 kDa and GHFPRAC, respectively, and corresponded to those of mature human tyrosinase. The RHT was active in a broad range of temperature and pH, and with optimum activity at 70 °C and pH 8.5.     

Die‐off of Escherichia coli in intact and disrupted cowpats

Diffuse pollution from grazed pastures is recognised as an important source of faecal microbial contamination of surface waters, and farmers urgently require mitigation options to reduce this impact of their farming activities. An obvious mitigation strategy is to reduce the size of the reservoir of faecal material in pastures during critical periods when overland flow is most likely. This work tested the hypothesis that smearing fresh cowpats to increase their surface area would increase exposure to solar radiation and increase desiccation, which in turn would accelerate the die‐off of Escherichia coli in the faecal material. The total solids content and E. coli concentration in intact and disrupted cowpats were monitored during the four seasons. The total solids content was significantly (P < 0.001) higher in the disrupted cowpats compared to the intact cowpats. This indicates that increased desiccation of the faecal material was successful. However, there was no significant difference (P > 0.05) in E. coli concentrations in the disrupted and intact cowpats. The physical disruption of cowpats did not therefore accelerate the die‐off of E. coli in the faecal material. The disrupted cowpats did appear to decompose faster than the intact cowpats. Mitigation options to reduce faecal micro‐organism losses in overland flow from grazed pastures will now need to focus on other methods to reduce the build up of reservoirs of faecal material on farm.     

重组甘蔗ACC氧化酶cDNA原核表达的纯化复性研究

重组甘蔗ACC氧化酶基因在大肠杆菌中表达,其产物以不溶性包涵体存在。用N i2 -NTA亲和层析柱对其进行纯化,结果显示,在层析柱上直接复性及纯化的方法比在变性条件下N i2 -NTA纯化然后稀释透析进行复性的方法简捷,效果好,获得的目的蛋白质纯度大于98%,活性为132.58 nm o l C2H4/(m g.h)。     

部分蜘蛛牵丝Ⅰ型蛋白基因的合成及其聚合体在大肠杆菌中的表达

已知蜘蛛牵丝Ⅰ型蛋白N端序列不完整，有大量多聚丙氨酸基序组成的重复区。根据已报道的金纺者（Nephila clavipes）相应基因序列，合成若干寡核苷酸链，通过PCR获得编码涵盖三组多聚丙氨酸基序的基因片断。并以此为单体，经过酶切和连接获得重复区分别为2、4、8拷贝的聚合体。序列及RNA二级结构比对显示：所获8聚体与已报道基因编码的氨基酸同源性高达84％，但8聚体基因mRNA具有较低能级。将上述3种聚合体基因分别克隆大肠杆菌（Escherichia coh）表达载体pET-30a（＋），经IPTG诱导、利福平阻断菌体基因表达及在培养基中添加^35S-Met，通过放射自显影可见由T7启动子引发的特异目的基因表达带。     

棉铃虫组织蛋白酶B酶原在大肠杆菌中的表达及纯化

用合成的基因特异性引物通过RT-PCR扩增，获得棉铃虫(Helicoverpa armigera)组织蛋白酶B基因(HCB)，将基因克隆到pGEX-4T-1载体，在大肠杆菌(Escherichia coli)DH5α内进行扩增，经过PCR筛选获得阳性克隆并提取质粒和测序验证后，再转化大肠杆菌BL21进行诱导表达。表达产物为组织蛋白酶B-谷胱甘肽S转移酶的融合蛋白，分子量在63kD左右。表达产物形成了包涵体，经过对包涵体变性和复性处理，得到了可溶性的融合蛋白，可被Glutathione Sepharose 4B柱亲和纯化，用凝血酶裂解融合蛋白后，经SDS-PAGE分离到37kD左右的棉铃虫组织蛋白酶B酶原。N-末端氨基酸测序鉴定表达产物为棉铃虫组织蛋白酶B。     

鸡MxA基因的克隆及其在大肠杆菌中的表达

MxA是由Ⅰ型干扰素诱导宿主细胞所产生的抗病毒蛋白家族的成员之一。采用RT-PCR方法从鸡胚成纤维细胞(CEF)中扩增MxA,将其克隆至载体pMD-T18中,筛选阳性克隆后回收目的片段,将其克隆入原核表达质粒pGEX-6p-1,构建其重组表达质粒pGEX-MxA,以IPTG诱导表达,经SDS-PAGE、鸡胚新城疫病毒增殖干扰实验和VSV-CEF微量细胞抑制实验进行分析、鉴定。结果表明,经RT-PCR扩增获得的MxA序列与GenBank报道的序列一致,SDS-PAGE和干扰实验证实重组质粒可以表达出相应分子量为45kD的蛋白,与GST-MxA融合蛋白分子量一致,影像分析系统分析显示表达的融合蛋白约占菌体蛋白的30%。     

Survival of Escherichia coli donor,recipient, and transconjugant cells in soil

Survival of Escherichia coli donor, recipient and transconjugant cells was studied in sterile soil incubated under a variety of conditions. In soil not amended with additional nutrients, the absence of transconjugants indicated that R-plasmid transfer had not occurred. In soil supplemented with a low level of nutrient broth, both donor and recipient cells survived over a 48 hr period. In addition, transconjugant cells were detected in soil as early as two hr after the soil was inoculated with donor and recipient strains.     

白颈长尾雉圈养种群大肠杆菌耐药性及其耐药基因

对白颈长尾雉圈养条件下的38个样品进行大肠杆菌分离及PCR检定,并采用肠杆菌基因间重复共有序列PCR指纹法（ERIC）剔除各个样品的重叠分离株,检测获得的170个大肠杆菌分离株对9种抗生素的耐药性、Ⅰ型整合子携带率及其可变区抗性基因,结果显示：（1）来自白颈长尾雉的分离株对实验用的9种抗生素的抗性比率和多重耐药性远高于环境源和人源者（来自白颈长尾雉的分离株100%耐受3种及以下的抗生素,而环境源者为50.7%,人源者66.7%）;（2）来自白颈长尾雉的分离株的Ⅰ型整合子携带率（92%）高于环境源（87%）和人源（78%）;（3）来自白颈长尾雉和来自人的大肠杆菌分离株的Ⅰ型整合子可变区抗生素抗性基因检出率相同（36%）,但高于环境源（24%）;（4）携带Ⅰ型整合子的分离株对实验用的抗生素的抗性百分率一般高于不携带者,只有个别种类抗生素这种差异为非显著性差异;（5）Ⅰ型整合子可变区基因盒的基因为3类,即aadA、dfrA和未知功能的orfF;aadA、dfrA的频率相同;3类基因均以基因盒形式存在,分别是dfrA17-aadA5、dfrA12-ofrF-aadA2、dfrA12-aadA2。