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。     

家蝇对氯菊酯抗性的分子机制研究

家蝇是一种重要的病媒生物,可以传播上百种人畜疾病。化学防治是家蝇控制的重要手段,但杀虫剂的重复和广泛使用可导致家蝇产生抗药性。在我国,不少事例显示家蝇对拟除虫菊酯杀虫剂普遍产生了抗药性,但对家蝇种群抗药性的分子机制还缺乏系统了解。本研究以来源于山东济南的家蝇为材料,在明确其对氯菊酯抗性水平的基础上,分析其抗药性的分子机制。结果显示,野外采集未经杀虫剂汰选实验室济南品系(JN)相对于敏感品系(WHO)表现出对氯菊酯近50倍的抗性,而经氯菊酯汰选7代后的JN-Sel品系获得了比JN更高水平的抗性,抗性倍数超过110倍。核酸检测显示JN和JN-Sel品系家蝇钠离子通道的基因型为1014LL敏感纯合型。通过检测8个细胞色素P450基因的表达水平,发现3个P450基因(CYP6A5v2、CYP6A36和CYP6G4)在抗性品系中的表达量高于敏感品系,其中CYP6G4的表达与氯菊酯抗性成正相关,JN品系CYP6G4的表达量是敏感品系的35倍,而在高抗性的JN-Sel品系中的表达量是敏感品系的70倍。研究结果强烈提示CYP6G4的过量表达是JN和JN-Sel品系对氯菊酯抗性的重要机制。     

抗药性与敏感性德国小蠊乙酰胆碱酶的活性比较

目的研究德国小蠊抗药性与乙酰胆碱酯酶活性的关系。方法采用化学比色方法对敏感品系和抗性品系的德国小蠊分别测试乙酰胆碱酯酶,对比两者的测定值,分析抗性品系和敏感品系德国小蠊乙酰胆碱酯酶的差异。结果敏感品系和抗性品系德国小蠊的乙酰胆碱酯酶活性分别是2.863和5.609,抗性品系德国小蠊酶活性显著高于敏感品系,两者酶活性比值为1.96。结论乙酰胆碱酯酶活性与德国小蠊抗药性有关。     

体内外光滑假丝酵母菌氟康唑耐药机制的比较研究

目的 研究比较临床分离和体外诱导的耐氟康唑光滑假丝酵母菌的耐药机制.方法 选取临床分离的4对氟康唑敏感-耐药配对的光滑假丝酵母菌,其中4株敏感株在体外氟康唑的作用下均被诱导为耐药株.利用罗丹明6G试验比较敏感株与两种耐药株的外排泵作用,实时荧光定量RT-PCR检测外排相关基因CDR1、CDR2、SNQ2和ERG11的表达.同时,对PDR1基因进行PCR扩增和测序,对比分析临床耐药株和体外诱导耐药株的突变位点.结果 临床耐药株和体外诱导耐药株外排泵的功能均明显强于敏感株；两者CDR1表达均显著升高,而CDR2和SNQ2则无明显变化；ERG11在敏感与耐药菌株之间的表达水平也无显著差别；两种耐药株的PDR1均发现错义突变位点,其中P927S、L543P及S947L突变尚未被报道过.结论 光滑假丝酵母菌在体内外氟康唑作用下PDR1均产生突变,引起外排相关基因,尤其是CDR1的表达增加从而增强了外排泵的作用导致耐药.     

北京地区德国小蠊(Blattela germanica)对氯菊酯抗性相关的钠通道基因点突变的研究

应用RT PCR扩增技术 ,克隆了北京地区自然种群德国小蠊 (Blattelagermanica)的钠离子通道基因片段 ,片段长度为 16 2bp ,并对其进行了测序。将推导的氨基酸序列与德国小蠊敏感品系的相应序列进行比较 ,发现在钠离子通道ⅡS4 ⅡS6的 993同源位点上存在Leu (TTG)→Phe (TTC)突变 ,表明该品系的抗药性可能与击倒抗性 (knockdownresistance ,kdr)有关     

志贺菌临床分离多重耐药株mar基因突变研究

目的 研究志贺菌mar基因突变与其多重耐药的关系.方法 对54株临床分离的志贺菌进行四环素、氯霉素、氨苄西林、环丙沙星、诺氟沙星等5种药物的药敏试验和有机溶剂耐受实、验(OST),对其,mar基因的marOR区进行聚合酶链反应-单链构象多态性分析(PCR-SSCP)及核酸序列分析.结果 筛选出35株多重耐药株,多重耐药率为64.8%.54株志贺菌株中有38株对有机溶剂耐受,其中有33株为多重耐药株,3株为单耐药株,仅有2株为敏感野生株.54株志贺菌临床分离株均扩增出MAR基因,未发现该基因缺失株,SSCP分析发现,35株多重耐药株中marOR基因突变率为14.29%.核酸测序分析发现志贺菌多重耐药株marO区基因1376-1379处4个碱基缺失,导致其后编码氨基酸出现移码突变,而标准株S51250和敏感野生株不存在该移码突变;marR区基因存在10处点突变,第1752碱基(G→A,Gly→Ser)和第1854碱基(T→c,Tyr→His)两处点突变导致编码氨基酸改变,但该突变也同时存在于标准株和敏感野生株中,余者均为无义突变,且有些核苷酸改变也发生于敏感野生株或同时存在于多株菌株中.结论 ,marO区基因突变可能是志贺菌多重耐药的调控机制之一.     

一种蟑螂抗性快速检测方法的探索

胶带或胶板用于蟑螂的监测已多年 ,本实验将其与药剂联合使用 ,将不同浓度的杀虫药剂涂于胶面上 ,将被试种群的德国小蠊粘于其上 ,2 4h后观察其死亡率。根据粘胶板上的不同药剂剂量 ,可以求出被试种群的死亡———剂量曲线 ,以实验室敏感种群的LC50 做为基数 ,便能够求出该种群的抗性指数。用该种方法对采自北京地区的 4个德国小蠊种群进行抗性检测 ,同时采用传统的药膜法对该 4个种群的击倒抗性进行测定 ,将此二者的结果进行比较 ,发现 4个种群的抗性大小的趋势基本一致。     

靶向阻断LRP提高肺腺癌细胞对紫杉醇的敏感性

目的利用小干扰RNA(siRNA)技术沉默肺腺癌细胞肺耐药相关蛋白基因(LRP),探讨其对肺腺癌紫杉醇耐药株(A549/TXL20)紫杉醇(TXL)敏感性的影响。方法逐步增加药物浓度法建立A549紫杉醇耐药细胞株(A549/TXL20),用小干扰RNA技术沉默LRP在A549/TXL20细胞中的表达,以MTT法检测紫杉醇对A549/TXL20细胞的半数抑制浓度(IC50);以q PCR检测细胞中LRP mRNA的表达,Western blot检测细胞中LRP蛋白的水平,裸鼠腋窝皮下注射转染后的A549/TXL20细胞,建立裸鼠移植瘤模型,观察LRP靶向siRNA对人肺腺癌耐药细胞株A549/TXL20移植瘤耐药的影响。结果肺腺癌紫杉醇耐药株(A549/TXL20)对紫杉醇的敏感性明显增强(P0.01),A549/TXL20细胞中LRP mRNA及蛋白表达显著增加(P0.01);siRNA沉默A549/TXL20细胞LRP基因后,与空白组和空质粒组比较,LRP mRNA及蛋白表达均被抑制(P0.01);小干扰RNA可提高荷瘤裸鼠对紫杉醇的敏感性。结论 siRNA可有效沉默A549/TXL20肺腺癌耐药细胞株LRP基因的表达,提高耐药的肺腺癌细胞对紫杉醇的敏感性。     

鲍曼不动杆菌临床株的外排泵研究

目的 探讨鲍曼不动杆菌临床株外排泵AdeABC、AdeIJK、AdeFGH、AbeM、AbeS、CraA、MdtL的表达与耐药的关系.方法 收集多重耐药鲍曼不动杆菌临床株32株和敏感株10株,PCR扩增泵基因;选取主要克隆型的21株多重耐药株和10株敏感株,实时荧光定量RT-PCR方法检测泵基因adeB、adeJ、adeG、abeM、abeS、craA、mdtL的mRNA相对表达水平,PCR扩增泵调控基因adeRS、adeL并测序分析.结果 32株多重耐药鲍曼不动杆菌临床株中携带泵结构基因片段adeB100％、adeJ 100％、adeG 100％、abeM 96.88％、abeS 100％、craA 100％、mdtL 93.75％,10株敏感株均存在7种泵结构基因片段;主要克隆型的21株多重耐药株和10株敏感株adeB、abeM、mdtL的mRNA相对表达水平的差异有统计学意义( P＜0.001,P=0.001,P=0.013),选取多重耐药株AbR3和AbR11检测外排泵AdeABC调控基因adeRS序列出现氨基酸替代及缺失,而外排泵AdeFGH调控基因adeL序列无基因突变.结论 鲍曼不动杆菌临床株外排泵AdeABC、AbeM、MdtL的表达可能与耐药性有关.     

Mechanisms underlying fipronil resistance in a multiresistant field strain of the German cockroach (Blattodea: Blattellidae)

German cockroaches (Blattella germanica L.) have significant impacts on human health, most notably they are implicated as causes of childhood asthma. Gel bait formulations of fipronil, a phenylpyrazole insecticide, have been in use for German cockroach control in the United States since 1998. Previously, dieldrin resistant German cockroach strains were shown to have 7- to 17-fold cross-resistance to fipronil. More recently, a field-collected strain (GNV-R) displayed approximately 36-fold resistance to topically applied fipronil at the LD50 level, which is the highest level of fipronil resistance reported to date in the German cockroach. The aim of the current research was to identify mechanism(s) responsible for high-level fipronil resistance in the GNV-R strain. Synergist bioassays conducted using topical and injection application methods implicated cytochrome P450-mediated detoxification in resistance. Electrophysiological recordings using the suction-electrode technique revealed the nervous system of the GNV-R strain is insensitive to fipronil. In agreement with electrophysiology results, the alanine to serine (A302S) mutation encoded by the gamma-amino butyric acid-gated chloride channel subunit gene resistance to dieldrin, which confers limited cross-resistance to fipronil, was detected in 95% of GNV-R strain individuals. Logistic regression analysis showed that A302S mutation frequency correlates with neurological insensitivity as shown by electrophysiology data. Overall, results of synergism bioassays, electrophysiological recordings, and A302S mutation frequency measurements suggest that fipronil resistance in the GNV-R strain is caused by the combined effects of enhanced metabolism by cytochrome P450s and target-site insensitivity caused by the A302S-encoding mutation in the resistance to dieldrin gene.     

CYP2C19、CYP3A5基因多态性与心肌梗死的相关性研究

目的探讨CYP2C19、CYP3A5基因的多态性与心肌梗死发病风险的相关性。方法随机选取心肌梗死患者及健康对照各500例,采用荧光PCR法和Sanger测序分别检测其CYP2C19、CYP3A5基因的多态性,用Logistic回归分析其与心肌梗死的相关性,用Quanto软件评估统计学效能。结果CYP2C19基因rs4986893位点的AG、GG基因型和A等位基因的频率以及CYP3A5基因rs776746位点的AA、AG、GG基因型和G等位基因频率在两组之间的差异具有统计学意义(P<0.05),CYP2C19基因rs4244285、rs12248560位点的基因型和等位基因以及rs4986893位点的AA基因型的频率在两组之间差异无统计学意义(P>0.05)。在校正年龄、性别、体质指数后,Logistic回归分析显示CYP2C19基因rs4986893的AG基因型和A等位基因以及CYP3A5基因rs776746的GG基因型和G等位基因可能是心肌梗死发病的风险因素,而rs4986893的GG基因型以及rs776746的AA、AG基因型可能是心肌梗死的保护因素。依据样本量、样本结构和等位基因频率以及Quanto分析,本研究的结果具有理想的统计学效能(99%)。结论CYP2C19、CYP3A5基因的多态性可能增加心肌梗死的发病风险。     

Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase.

A series of Salmonella typhimurium (S. typhimurium) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase (OR), was established. The parental S. typhimurium YG7108, derived from TA1535, lacks two O(6)-methylguanine-DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents. The expression levels of CYP holo-protein in the genetically engineered S. typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4. The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture. Each form of CYP expressed in the S. typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate. The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. These S. typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved.     

家蝇抗药性的分子遗传机制

综述了近年来家蝇抗药性分子机制的研究进展。脂族酯酶基因(MdaE7)在位置137(Gly)和251(Trp)的氨基酸突变可能与家蝇对有机磷抗性有关;谷胱苷肽S转移酶(GSTs)超量表达在家蝇对有机磷农药的抗性中起重要作用;有机磷抗性家蝇的AChE基因普遍存在Gly262Ala和Phe327Tyr突变。击倒抗性(kdr)和P450介导的代谢抗性是家蝇对DDT和拟除虫菊酯类杀虫剂抗性的重要机制,电压门控钠离子通道的L1041F突变产生Kdr,而M918T和L1041F双突变导致超击倒抗性(superkdr);P450介导的代谢抗性表现出进化可塑性的特点。有关家蝇抗性机制的阐明,可为抗性的检测和监测提供准确和方便的手段。     

Sensitive developmental period of last-instar German cockroaches (Dictyoptera: Blattelidae) to fenoxycarb and hydroprene

The sensitive developmental period of last-instar German cockroaches, Blattella germanica (L.), to topical applications (10 micrograms/microliters) of fenoxycarb and hydroprene was determined. Developmental duration of last instars treated with fenoxycarb on days 1, 3, or 6 was 9-11 d longer than the development of nymphs treated with hydroprene or acetone. In addition, fenoxycarb caused mortality in 59% of the nymphs treated on day 6. A high level of wing twisting and sterility was observed among female and male cockroaches treated with fenoxycarb or hydroprene on days 1, 3, or 6 of the last stadium. Nymphal production by adults treated with juvenoids on day 9 of the last stadium (3-4 d before eclosion) was not affected. Our findings show that the sensitive developmental period for juvenoid-induced effects in last-instar B. germanica is limited to the first half of the stadium.