EXPRESSION OF cDNA FOR RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR IN ESCHERICHIA COLI AND CHARACTERIZATION OF THE PROTEIN

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Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)—what is the difference?

Two forms of recombinant human G-CSF (rhG-CSF) are available for clinical use: filgrastim is expressed inE coli and non-glycosylated, whereas lenograstim is derived from Chinese hamster ovary (CHO) cells and glycosylated. The function of the sugar chain, accounting for approximately 4% of the molecular weight of lenograstim (and native G-CSF), is not known. Glycosylation of the G-CSF molecule does not prolong its circulation half life. Lenograstim is more active than filgrastim (and research-use deglycosylated G-CSF) on a weight-by-weight basis inin vitro colony-forming and cell line assays. An international potency standard assigns a specific activity of 100 000 IU/μg to filgrastim and 127 760 IU/μg to lenograstim. Correspondingly, two randomised crossover studies in normal subjects, comparingmass equivalent doses of the two rhG-CSFs, have demonstrated a 25–30% higher concentration of blood stem cells (CD34⁺, CFU-GM) during lenograstim administration. No difference in side effects was observed. Results from a prospective, randomised, non-crossover trial in breast cancer patients suggest thatbioequivalent doses of filgrastim and lenograstim have a similar effect on mobilisation of CD34⁺ cells and immature CD34⁺ cell subsets, respectively. Although comparisons outside the setting of stem cell mobilisation are lacking, the clinical relevance of the greater specific activity of lenograstim may thus be limited. The difference in potency between μg identical doses of the two rhG-CSFs makes dosing in biological units (IU) rather than mass units (μg) more appropriate.     

G-CSF在人实体肿瘤细胞株中的表达及其对细胞增殖的影响

目的:探讨G-CSF在人实体肿瘤细胞株中的表达及外源性rhG-CSF对肿瘤细胞增殖的影响及机制。方法:人G-CSF ELISA试剂盒检测肿瘤细胞G-CSF的分泌水平,细胞计数法和MTT法检测rhG-CSF对肿瘤细胞增殖的影响,流式细胞仪分析rhG-CSF对KB细胞周期和细胞凋亡的影响,免疫细胞化学检测G-CSF受体在肿瘤细胞中的表达。结果:T-24细胞分泌大量G-CSF;rhG-CSF呈剂量依赖性促KB细胞增殖,并在质量浓度为100 ng/mL达最大值,与对照相比,差异有统计学意义(P<0.05);KB细胞表达G-CSF受体,rhG-CSF拮抗KB细胞的G0/G1期阻滞,降低KB细胞凋亡率。结论:T-24细胞表达G-CSF;rhG-CSF通过作用于KB细胞表面的G-CSF受体拮抗G0/G1期阻滞、抑制凋亡而发挥促增殖作用。     

死亡受体5胞外区域的重组、表达及活性鉴定

目的: 构建死亡受体5(death receptor 5,DR5)胞外区域(eDR5)的表达载体,表达纯化重组蛋白并鉴定其生物特性.方法: 通过重叠 PCR 获得 DR5 胞外段编码序列,构建 pET-22b( )/DR5 表达载体,转化大肠杆菌 BL21(DE3),IPTG 诱导表达,Ni2 柱亲和纯化,SDS-PAGE、直接 ELISA 鉴定纯化产物的纯度和特异性,用 MTT 法检测eDR5 蛋白阻断DR5 单克隆抗体 FMU1.5 和 TRAIL 诱导人胶质瘤细胞株 U343(高表达DR5)、U373(低表达DR5 )细胞凋亡的作用.结果: 获得了 DR5 胞外段编码序列,目的蛋白在上清及包涵体中都有表达, 表达量占菌体总蛋白的 30% 以上,纯化的重组蛋白纯度达 95% 以上,蛋白产量达 9 mg/ml.ELISA 结果表明所纯化蛋白为eDR5.eDR5 蛋白可部分阻断 FMU1.5 和 TRAIL 诱导人胶质瘤细胞株 U343 细胞凋亡的作用,其阻断率与 DR5 表达相关.结论: 死亡受体 5 胞外段基因的成功重组、表达及纯化,为进一步的功能研究奠定了基础.     

rhG-CSF affects genes involved in mitogen signalling and early gene expression in the ovarian cancer cell line HEY

The ovarian adenocarcinoma cell line HEY was used as an in vitro model to study the influence of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on epithelial tumours such as ovarian cancer. Serum-starved cells were treated with rhG-CSF in a time- and dose-dependent manner. Cell proliferation, measured as cell division and DNA synthesis, was stimulated about 40% by rhG-CSF. After harvesting, cells were examined for the presence of G-CSF receptor (FACS analysis and RT-PCR), as well as for expression of genes involved in mitogen signalling (ERKs, JNKs) and early gene expression (c-jun). rhG-CSF affected mitogen-activated pathways and was receptor-mediated if the G-CSF receptor was present. After rhG-CSF induction, Janus N-terminal kinases (JNK 1 and 2) were simultaneously increased in the cytosol, up to 30-fold as measured by Western blotting), whereas ERK 1 and 2 accumulated maximally by 2.5-fold 1 hr after rhG-CSF induction. c-Jun was up-regulated strongly by this cytokine at the translational level. Our data suggest that rhG-CSF affects genes involved in mitogen signalling and early gene expression in solid tumours. We also noted the presence of G-CSF receptor on ovarian cancer cell lines. Int. J. Cancer 75:847–854, 1998. © 1998 Wiley-Liss, Inc.     

EGF-Ang融合蛋白的构建、表达及活性研究

目的：构建由人表皮生长因子（epidermal growth factor,EGF）和人血管生成素（angiogenin, Ang）组成的人源化的融合蛋白，并检测其对肿瘤细胞的靶向杀伤能力。方法：利用基因工程技术将EGF和Ang基因连接起来，克隆到高效表达载体pET28a(+)中，构建重组表达质粒pEGFAng，并在大肠杆菌中表达该融合蛋白（EGFAng）。经DEAESepharose FF阴离子交换柱层析纯化后，用MTT法检测复性蛋白的细胞毒性。结果：SDSPAGE和薄层扫描分析表明外源蛋白的表达量占菌体裂解蛋白总量的18.6%。细胞活性检测表明EGFAng重组蛋白能明显地抑制Hep2细胞的生长，而不影响MA104细胞的正常生长。结论：融合蛋白EGFAng在体外对过度表达EGFR的Hep2细胞具有明显的杀伤作用。     

人截短型线粒体凋亡诱导因子蛋白的克隆、表达及诱导肝癌细胞凋亡的活性鉴定

背景与目的:线粒体在细胞凋亡中扮演关键的角色,线粒体凋亡诱导因子(apoptosis-inducing factor,AIF)是定位于线粒体中的一种重要的凋亡蛋白,对线粒体蛋白质的研究可深入阐明线粒体在凋亡中的作用.本研究的目的在于克隆、表达重组的人截短型AIF,并对其诱导肿瘤细胞核凋亡的生物学活性进行鉴定.方法:采用RT-PCR技术从人肝癌细胞SMMC-7721中扩增出剪切掉线粒体定位信号的人AIF基因片段,并按阅读框克隆到原核表达载体pET32a( )中.进行酶切与测序鉴定后,以构建的正确重组质粒pET32a-AIF转化大肠杆菌BL21(DE3)菌株,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达AIF蛋白,表达产物用SDS-PAGE和Western blot检测;采用镍柱亲和层析法纯化目的蛋白;采用凝胶滞后实验(EMSA)与Hoechst 33258染色检测AIF蛋白的生物学活性.结果:获得了去掉线粒体定位信号的AIF基因,并克隆到pET32a( )载体中,经酶切与测序鉴定完全正确.此重组质粒转化人大肠杆菌,经IPTG诱导后,在大肠杆菌中可表达相对分子质量约Mr 70 000的目的蛋白;表达量约占菌体蛋白总量的11%,表达的目的蛋白与抗His标签抗体与抗人的AIF蛋白具有良好的反应性;纯化后,AIF的纯度达到95%.经EMSA与Hoechst 33258染色实验证实:获得的AIF蛋白具有良好地与DNA结合并可诱导肿瘤细胞核凋亡的能力,凋亡率为37%.结论:人AIF基因在PET表达系统中得到有效表达:蛋白复性后能有效地在体外与DNA结合,并可诱导细胞凋亡.     

Pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol in rats.

The pharmacokinetics of recombinant human granulocyte colony-stimulating factor conjugated to polyethylene glycol (PEG-rhG-CSF) and rhG-CSF were studied in male Sprague-Dawley rats. The serum concentration after i.v. administration at a dose of 100 micrograms protein/kg was investigated by a bioassay. The serum rhG-CSF concentration decreased steadily after injection with a terminal half-life of 1.79 h. The PEG-rhG-CSF concentration after injection decreased much more slowly with a half-life of 7.05 h. The slower disappearance of PEG-rhG-CSF resulted in a greater area under the concentration-time curve. The neutrophil count after 100 micrograms of protein/kg of rhG-CSF administration reached a peak 12 h after injection and returned to the control level 48 h after injection. The neutrophil count after 100 micrograms of protein/kg of PEG-rhG-CSF administration was identical to that of rhG-CSF after 12 h but the highest level was maintained for 24 to 72 h after injection and returned to the control level after 168 h. These data indicated that PEG-rhG-CSF administration exerted a sustained biological effect on peripheral blood neutrophils. It is expected that PEG-rhG-CSF may contribute greatly to human G-CSF treatment because it has a prolonged neutrophil-proliferating activity enabling fewer administrations.     

血管形成抑制因子HIAF-1在昆虫细胞中的高效表达和活性研究

背景与目的：大肠杆菌表达的人抑制血管生成因子－1（human inhibiting angiogenesis factor-1,HIAF-1）可抑制肿瘤血管的形成。本研究在昆虫细胞中表达HIAF－1,并研究其生物活性。方法：用DNA重组技术,构建了重组杆状病毒表达载体pAcuW51-HIAF-1,用Cellfectin将其与线性病毒DNA共转染昆虫细胞sf9,获得重组病毒。用SDS－PAGE电泳和Western blot鉴定重组病毒感染的昆虫细胞中重组HIAF－1重组蛋白的表达。重组蛋白经亲和层析纯化后,在体外用MTT法检测其对内皮细胞的作用,用食管癌移植瘤研究其体内抑瘤活性。结果：在昆虫细胞中高效表达了分子量为26kDa的HIAF－1重组蛋白,其表达量可达昆虫可溶性蛋白的5％－10％。重组HIAF－1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3．1μg/ml,而且显著抑制食管癌移植瘤的生长。结论：在昆虫细胞中高效表达了分子量为26kDa的HIAF－1重组蛋白,其表达量可达昆虫可溶性蛋白的5％－10％。重组HIAF－1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3．1μg/ml,而且显著抑制食管癌移植瘤的生长。结论：在昆虫细胞中高效表达了HIAF－1重组蛋白;HIAF－1重组蛋白的生物活性优于相应的原核重组蛋白。     

牛蛙核糖核酸酶在大肠杆菌中的高效表达及其初步纯化

目的:利用大肠杆菌系统表达重组RC蛳RNase融合蛋白,并对其表达产物进行初步纯化。方法:以PCR方法扩增出RC蛳RNase基因,插入到融合蛋白原核表达载体PET32a中,构建重组表达载体PET蛳RC蛳RNase,转化大肠杆菌E.coliBL21(DE3)plyS,利用异丙基硫代蛳β蛳D蛳半乳糖苷(IPTG)诱导表达。工程菌经超声碎菌,离心后,采用Ni亲合层析法在变性条件下进行初步纯化。结果:经IPTG诱导后,SDS蛳PAGE和免疫印迹分析显示,工程菌在相对分子质量为3.1×104处出现一条新生蛋白带,目的蛋白占菌体总蛋白的34%,主要以包涵体形式存在。所得包涵体经纯化,纯度可达90%以上。结论:重组融合蛋白RC蛳RNase在大肠杆菌中获得成功表达,纯化效果令人满意,为进一步研究其生物学特性和功能奠定了良好的基础。     

Sub-MIC Ciprofloxacin Effect on Fimbrial Production by Uropathogenic Escherichia coli Strains

Abstract

The urine from 210 patients with acute urinary tract infection (UTI) was examined to study the in vitro effect of ciprofloxacin on fimbriae production by uropathogenic Escherichia coli isolates. Forty-nine bacterial samples of density 105 CFU/ml were not considered. From the resulting 161 samples, E. coli was the major strain found, present in 54 samples. Other microoganisms found were: Enterococcus sp. (34 samples), Staphylococcus epidermis (22), yeasts (11), Proteus sp. (11), Pseudomonas sp. (11), Klebsiella sp. (8), Enterobacter sp. (6), Citrobacter sp. (3), and Acinetobacter sp. (1).

The uropathogenic E. coli strains found were P-fimbriated, as demonstrated by hemoagglutination activity against human erythrocytes with and without mannose, SDS-PAGE of fimbrial proteins and transmission electron microscopy (TEM).

All E. coli strains found were exposed in vitro to sub-inhibitory concentrations of ciprofloxacin (1/8 MIC). Our results showed that: 1) P-fimbriated E. coli is the most prevalent microorganism in acute UTI (34%); 2) exposure to sub-MICs of ciprofloxacin inhibits fimbrial production in 79% of E. coli strains; 3) the pattern of SDS-PAGE fimbrial proteins is modified after exposure; in particular, the most affected synthesis involves the protein at 18 kD known as P-fimbriae.     

Diphtheria toxin/human B-Cell activating factor fusion protein kills human acute lymphoblastic leukemia BALL-1 cells: An experimental study

Objective: This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). Methods: A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni2+-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion: The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.     

细胞凋亡相关新基因Apr-3的一个转录剪接体的克隆、初步表达及纯化

目的克隆Apr-3基因的一个转录剪接体,进行原核表达,并进行初步纯化。方法培养HL-60细胞,提取HL-60细胞总RNA,应用RT-PCR获取Apr-3编码区cDNA序列的前366bp,将该cDNA与质粒pcDNA3.0连接,构建克隆载体,将其转化E.coliDH5α,进行测序,与GenBank中登录序列比对,结果正确后将该cDNA与质粒pET28a连接,构建原核表达载体,转化大肠杆菌,诱导表达获得Apr-3蛋白。结果对测序结果进行序列分析,与GenBank中登录的Apr-3编码区完全一致。Apr-3融合蛋白主要以包涵体形式存在。结论成功构建了Apr-3的原核表达载体,获得了较纯的Apr-3融合蛋白。     

靶向白血病干细胞CD123的毒性融合蛋白的制备

 目的　构建靶向白血病干细胞CD123的融合蛋白,检测其表达效果及其识别白血病干细胞的活性。方法　采用PCR法扩增白细胞介素-3（IL-3）和毒素蛋白（LP）的编码DNA序列,经酶切、连接,克隆至表达载体pAYZ,转化大肠杆菌E.coli 16C9,鉴定阳性克隆菌落,经低磷培养基AP5诱导表达融合蛋白IL-3-G4S-LP,用CM-FF阳离子柱和抗-Etag亲和层析柱纯化,纯化产物经SDS-PAGE和Western blot鉴定,流式细胞术测定其与红白血病细胞TF1的结合活性。结果　构建的融合蛋白在大肠杆菌中以可溶性蛋白表达,纯化后纯度达95 %以上,表达量约为1 mg／L,并有与白血病干细胞表面IL-3受体α亚基（CD123）特异性结合的活性。结论　构建的融合蛋白IL-3-G4S-LP具有与白血病干细胞结合的特性,可望成为复发和耐药白血病的治疗药物。     

骨肉瘤细胞(MG-63)条件培养基中骨形态蛋白(BMP)的实验研究--BMP的分离、纯化、鉴定

目的 探索从骨肉瘤细胞条件培养基中提取骨形态蛋白(BMP)的方法，并测定其生物学活性。方法 收集骨肉瘤细胞(MG-63)条件培养基，通过浓缩、透析，SephcrylS—100凝胶层析纯化，BMP单克隆抗体鉴定所需洗脱峰，SDS—PAGE测定分子量，小鼠肌袋实验检测其骨诱导活性。结果 BMP单抗鉴定所提蛋白为BMP，SDS—PAGE显示分子量在21kD，能够在小鼠肌肉内产生异位骨化。结论 骨肉瘤细胞条件培养基中含有BMP，分离后具有良好的生物学活性，而骨肉瘤细胞可以在体外长期培养生长，为BMP的大量提取、临床应用提供一个有益的方法。     

抗人肺腺癌单链抗体的构建及在大肠杆菌中的表达

目的:构建抗肺腺癌的单链抗体(scFv),研究其表达条件,为将这一小分子抗体应用于临床奠定基础.方法:将抗人肺腺癌单克隆抗体的重链及轻链可变区基因插入表达载体pFUW80、pTH、pTF,分别在大肠杆菌XL1-Blue、Top10、GI698/GI724中诱导表达,得到噬菌体抗体或包涵体.用SDS-PAGE和ELISA鉴定检测活性.结果:SDS-PAGE的结果表明,在pTH和pTF的表达产物中,有一条43kD的特异条带,其分子量与预期相符,单链抗体以包涵体形式出现,其表达量达细菌总蛋白的18.6%.ELISA的结果表明,噬菌体抗体和经复性处理的包涵体具有与亲本单抗相同的特异性.结论:成功地构建和在大肠杆菌中表达了抗肺腺癌单链抗体.