德国小蠊细胞色素P450 CYP4G19基因的原核表达及多克隆抗体制备

目的构建细胞色素P450 CYP4G19基因部分片段的原核表达载体并诱导其表达,纯化表达的融合蛋白并制备CYP4G19多克隆抗体。方法应用RT-PCR扩增CYP4G19基因部分片段,产物经T-A克隆、测序鉴定,亚克隆入原核表达载体pET-28a,在大肠杆菌BL21（DE3）中诱导表达,镍离子亲和层析法纯化重组蛋白后,免疫小鼠,获得多克隆抗体,ELISA及Western-blot检测多抗的效价及特异性。结果从德国小蠊cDNA中克隆出一段771bp的亲水性基因片段,在大肠杆菌中诱导表达出约32000Mr、以包涵体形式存在的P450重组蛋白。将纯化、复性的重组蛋白免疫小鼠。得到了滴度高于1：10^6的高效价多克隆抗体。Western-blot显示此多抗能与32000Mr的重组蛋白特异结合,并能识别天然的德国小蠊微粒体P450蛋白。结论利用原核表达的CYP4G19融合蛋白具有良好的免疫原性。制备出效价高、特异性强的抗德国小蠊CYP4G19多克隆抗体,为下一步关于德国小蠊CYP4G19蛋白表达特性及其抗药性功能的深入研究提供了重要的实验工具。     

Cytochrome P450 expression is a common molecular event in soft tissue sarcomas

Two major xenobiotic metabolizing sub-families of cytochrome P450 (cytochrome P450 1A and cytochrome P450 3A) have been identified in soft tissue sarcomas. Cytochrome P450 1A was present in 70 per cent and cytochrome P450 3A was present in 78 per cent of tumours, respectively. A high proportion (86 per cent) of those tumours which contained cytochrome P450 1A or cytochrome P450 3A demonstrated co-expression of both sub-families. In each tumour, cytochrome P450 immunoreactivity was identified in all tumour cells and there was no intra-tumour heterogeneity. These results indicate that expression of cytochrome P450 is a common molecular event in soft tissue sarcomas and that the presence of different sub-families of cytochrome P450 has implications both for the pathogenesis and for the treatment of these tumours. Cytochrome P450 expression may influence the intrinsic drug resistance of these tumours and also provide a molecular target for anti-cancer drugs which can be activated by cytochrome P450.     

细胞色素P450基因多态性与药物代谢

细胞色素P450(cytochrome P450,CYP)在众多外源性和内源性物质的代谢中具有重要作用.CYP家族1-3中编码P450的基因均存在多态性,特别是CYP2C9、CYP2C19、CYP2D6和CYP3A5.超过一半的临床药物是由多态性P450介导代谢,CYP基因的多态性是造成药物反应个体差异的主要原因.近几年,许多与P450酶活性和CYP基因表达相关的等位基因已被鉴定,因此通过分型CYP基因的功能性或标签(Tag)的遗传变异,就可以获得个体的代谢表型,有助于医生及时找到正确的用药方案,有效地提高药物疗效和降低毒副作用,特别是那些治疗指数窄的药物.显然,了解CYP基因的遗传变异对于临床药物治疗和药物开发是必不可少的.基因芯片技术具有高多重水平和高通量的特点,使同时分型大量CYP基因遗传变异成为可能,是实现个性化医疗的重要技术保障.然而,DNA制备制约了预测性CYP基因分型芯片的发展,其在临床上的广泛应用尚需时日.     

人源细胞色素C在大肠杆菌中的重组表达和分离纯化

细胞色素C在电子传递、缺氧应激和细胞凋亡的过程中起重要作用。通过人源细胞色素C和酵母细胞色素C亚铁血红素裂解酶共表达,从大肠杆菌中获得了可溶性的重组人源细胞色素C蛋白。细菌经过超声破碎后,获得的上清经350g/L硫酸铵沉淀去除杂蛋白,再经过两次SP-Sepharose Fast Flow阳离子交换层析,获得了纯度大约为80%的人源细胞色素C。每升菌可产出10mg以上重组细胞色素C。人源细胞色素C的重组表达和纯化有助于细胞色素C功能的深入研究和应用。     

Cytochrome P450 gender-related differences in response to hyperoxia in young CBA mice

Cytochrome P450 monooxygenases (CYPs) represent large class of heme-containing enzymes that catalyze the metabolism of various endogenous and exogenous substrates. Although they are found in many tissues, the function of the particular subset of their isoforms does not appear to be the same. Many CYP genes exhibit sexually dimorphic expression, while others are sex-independent. Moreover, as a source of reactive oxygen species (ROS), P450 system is believed to play the important role in various pathological conditions and diseases. The aim of this study was to observe the effect of hyperoxia on oxidant/antioxidant status in the liver of young male and female mice and to determine whether the observed effects are associated with the expression of Heme oxygenase-1 (HO-1) and CYP genes associated with stress (Cyp1a1, Cyp1a2, Cyp2a5, and Cyp2e1) or stress and gender-related responses (Cyp2b9). In this study, we demonstrated gender-related effect of hyperoxia on oxidant/antioxidant status and on expression of certain P450 enzymes. Our results suggest that females are less susceptible to hyperoxia induced oxidative stress by two major mechanisms: upregulated expression of HO-1 genes and different expression of certain P450 enzymes. Therefore, our study could provide additional data of gender-dependent responses in susceptibility to oxidative stress, chemical toxicity and drug efficiency in treatment of diseases.     

生长激素对大鼠肾脏微粒体细胞色素P4504A2的影响

用Ｗｅｓｔｅｒｎ免疫印迹技术检测生长激素对大鼠肾脏微粒体细胞色素Ｐ４５０４Ａ２的影响。大鼠肾微粒体细胞色素Ｐ４５０与肝脏的一样，有种属、性别差异特异性。正常雄必大鼠肾微粒细胞色素Ｐ４５０４Ａ２含量大量于雌性大鼠的含量。切除长体的雄性和雌性大鼠肾微粒体细胞色素Ｐ４５０４Ａ３含量明显高于正常对照大鼠的水平，而雌、雄性之间的差异已不明显。用谷氨酸钠盐（ＭＳＧ）损伤雌、雄大鼠，雄性大鼠肾Ｐ４５０４Ａ２水平     

肝富集的转录因子与细胞色素P450基因的表达调控

细胞色素P450(cytochromeP450,CYP)是含血红素酶家族,广泛存在于从细菌到哺乳动物中,参与氧化或还原代谢多种内源和外源化合物。如内源性甾体激素、维生素和脂肪酸衍生物的合成与分解,外源性药物、杀虫剂、环境污染物和致癌物的代谢[1]。CYP1-4家族主要代谢外源化合物,它们表现     

Use of Drugs that Act on the Cytochrome P450 System in the Elderly

OBJECTIVES:

The objective of this study was to analyze medications that act on the cytochrome P450 (CYP450) enzymatic system and are used daily by non-institutionalized elderly individuals.

METHODS:

A cross-sectional population-based study of elderly individuals (≥ 60 years old) was conducted. All continuously used medications with hepatic metabolism via CYP450 that are classified as substrates, inducers or inhibitors were considered. For the analysis, elderly individuals were stratified according to age groups, and hepatic metabolism activity due to daily alcohol consumption and smoking were considered.

RESULTS:

Elderly individuals (396 in total: 222 women and 174 men) between 60 and 95 years of age (mean: 72.1) were assessed. Use of drugs that act on CYP450 was identified in 61.6% of the subjects. Drug use was observed among 16.2% of the subjects: three drugs among 9.8% and four or more among 6.3% of the subjects. The metabolic activities of the drugs used were classified as substrates (58.8%), inhibitors (14.9%), and inducers (4.3%). The main drugs used were beta-blockers and statins (as substrates), proton pump inhibitors and fluoxetine (as inhibitors), and prednisone and carbamazepine (as inducers).

CONCLUSIONS:

The results demonstrate that the elderly use high levels of medications that act on CYP450, thereby increasing the risk of drug interactions in a group that is already vulnerable to adverse drug effects.     

细胞色素P450与肿瘤的基因治疗

利用细胞色素P450治疗肿瘤是一种新的基因治疗方法,对提高肿瘤化疗的安全性和有效性有非常大的潜力。这种方法的首要目标就是选择性地使细胞色素P450在肿瘤细胞内超量表达。P450酶系降解的前药大部分在肿瘤内活化,提高了肿瘤细胞内药物的相对浓度,减小药物对其它组织的细胞毒性。本文概述了可被细胞色素P450降解的用于肿瘤治疗的前药以及重组细胞色素P450在基因治疗中的应用。     

小鼠SAP基因的克隆、表达及多抗制备

目的 制备小鼠SAP多克隆抗体,为进一步研究其功能和探讨其与炎症,肿瘤等疾病的相关性提供素材。方法 PCR扩增小鼠SAP基因编码区的DNA片断,将其重组入原核表达质粒pET30a(+),转化入大肠杆菌BL21中,异丙基β-D硫代办乳糖苷（IPTG）诱导产生His/mSAP融合蛋白,SDS-PAGE分析结果表明,蛋白主要以包涵体形式存在。采用割胶回收的方法纯化目的蛋白,免疫新西兰白兔,制备抗血清。通过ELISA、Western blot来检测抗血清效价及特异性。结果 成功的构建了pET30a(+)/mSAP重组质粒,表达融合蛋白,免疫兔子所获的mSAP多克隆抗体。结论 mSAP多克隆抗体特异性强,效价高。     

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.     

广州地区汉族人群细胞色素P450 1A1和细胞色素P450 2E1基因多态性

目的 探讨广州地区汉族人群细胞色素Ｐ４５０ １Ａ１（ＣＹＰ１Ａ１）和细胞色素Ｐ４５０ ２Ｅ１（ＣＹＰ２Ｅ１）基因的多态性分布规律。方法 用ＰＣＲ－ＲＦＬＰ和等位基因特异性扩增技术,对１５０名广州地区汉族正常人的ＣＹＰ１Ａ１和ＣＹＰ２Ｅ１基因多态性进行了检测,并与其他人群进行了比较。结果 ＣＹＰ１Ａ１基因３’端非翻译区的Ｍｓｐ１多态位点ｍ１（ＭＳＰⅠ－）、ｍ２（ＭｓｐⅠ＋）等位基因频率分别为６２．３     

我国汉族人CYP2C9新基因型的克隆及其在大肠杆菌中的表达

本研究采用RT-PCR方法成功的扩增到了细胞色素P450 2C9基因。测序分析结果表明,除编码区第190位核苷酸发生G→T的突变外,其它核苷酸序列与GenBank中登录发表的人P450 2C9基因(NM_017460)完全相同。SDS-PAGE和Western blot分析结果表明,经连接到pET-28a(+)原核表达载体后,CYP2C9基因在BL21(DE3)大肠杆菌中进行了成功表达,这为建立我国不同种族人群特有药物代谢酶药物评价体系打下了基础。     

Expression of selected cytochrome P450 isoforms and of cooperating enzymes in colorectal tissues in selected pathological conditions

The current interest in CYP expression in the colon results from its uniqueness as a target organ for cancer.     

Nono蛋白的原核表达、纯化和多克隆抗体的制备

目的表达和纯化带多聚组氨酸(6×His)标签的Nono(non-POU-domain-containing,octamer-bindingprotein)融合蛋白并制备抗Nono多克隆抗体。方法构建pET-28a( )-Nono重组表达质粒,转入Rosetta(DE3)大肠埃希菌,以IPTG诱导6×His-Nono融合蛋白表达,经镍离子金属螯合树脂纯化后,用纯化出的蛋白免疫BALB/C小鼠制备多克隆抗体,并用ELISA检测多克隆抗体的效价,Western印迹检测多克隆抗体的特异性。结果在大肠埃希菌中诱导出高水平表达的His-Nono融合蛋白,经亲和树脂纯化后免疫小鼠,获得了高特异性的抗Nono抗血清。结论成功构建pET-28a( )-Nono原核表达质粒,表达并纯化出高纯度的目标蛋白,制备出高滴度、高特异性的多克隆抗体。     

细胞色素P450 1A1在自然流产患者胎盘绒毛组织中的表达及意义

目的　探讨细胞色素P4 5 0 1A1(CYP1A1)mRNA在自然流产患者胎盘绒毛组织中的表达及其意义。方法　采用半定量逆转录聚合酶链反应 (RT -PCR)法检测 4 5例自然流产患者 (研究组 )中及 30例正常妊娠孕妇 (对照组 )胎盘绒毛组织中CYP1A1mRNA的表达。结果　自然流产及正常妊娠胎盘绒毛组织中CYP1A1mRNA相对表达强度分别为 0 .5 6± 0 .2 0、0 .35± 0 .14、两组比较差异显著 (P <0 .0 1)。自然流产患者中孕期有被动吸烟及孕期有饮茶或咖啡习惯者的胎盘绒毛组织中CYP1A1mRNA表达最强。结论　胎盘绒毛组织中CYP1A1mRNA表达增强可能与自然流产的发生相关 ,同时可在一定程度上说明孕妇吸烟、被动吸烟及摄入过多咖啡因可通过增强胎盘绒毛组织中CYP1A1的转录水平而增加自然流产的风险。     

中国汉族人细胞色素P450 2D6Ch基因特点研究

细胞色素Ｐ４５０２Ｄ６（ＣＹＰ２Ｄ６）基因的多态性是一种药物代谢的遗传变异，其表型分为强代谢型和弱代谢型。而且表型和基因的多态性存在明显的种族差异。本文利用ＰＣＲ－ＲＦＬＰ技术对１００名正常中国人ＣＹＰ２Ｄ６基因的Ｃｈ型突变特点做了分析，结果发现，在ＣＹＰ２Ｄ６基因的第１８８位点Ｃ→Ｔ的频率为０．５７，第４２６８位点Ｇ→Ｃ的频率为０．６６，而且Ｔ１８８／Ｔ１８８、Ｔ２９８８／Ｔ２９８８及Ｃ４２６８／Ｃ４２６８纯合子的比率分别为０．３２、０．０８和０．４２。与国外报道比较，发现中国人ＣＹＰ２Ｄ６基因的这两个位点的多态性分布规律与西方人有明显的不同。     

结核分枝杆菌Rv0073基因在大肠杆菌中的表达及纯化

目的 构建结核分枝杆菌(MTB) Rv0073基因原核表达载体并进行表达和纯化.方法 以MTB H37Rv基因组DNA为模板,采用聚合酶链反应(PCR)扩增目的基因片段,构建原核表达载体pET26b-Rv0073,经测序确定无误后转化至大肠杆菌(E.coli)感受态细胞BL21中.用聚丙烯酰氨凝胶电泳(SDS-PAGE)方法检测重组蛋白表达,检测异丙基-β-D-硫代半乳糖苷(IPTG)诱导不同时间、不同温度条件下重组蛋白表达量.采用His镍磁珠进行外源蛋白小量纯化.结果 成功构建重组表达质粒,重组蛋白经IPTG诱导后,2h开始明显表达且表达量无时间依赖性,在不同温度诱导下,重组蛋白的表达量随温度的增高而减少.重组蛋白以包涵体形式存在,经His镍磁珠纯化后获得重组蛋白.结论 成功构建并表达Rv0073蛋白,为后续Rv0073的大量纯化及其功能研究奠定了基础.     

脊椎动物P450s超家族在转基因动物中的研究进展

细胞色素P450s超家族参与生物体内许多物质的代谢反应。现就P450s家族的概况，人P450s家族的分类和主要功能，以及斑马鱼P450s的研究现状作一综述。同时介绍了应用转基因小鼠研究人P450s功能的进展，并对应用转基因斑马鱼研究人P450s在药物代谢方面的作用作一展望。