Detection of BCR-ABL fusion gene by multiplex RT-PCR combining with capillary electrophoresis

目的建立多重逆转录-聚合酶链反应(rT-PCr)联合毛细管电泳法检测bCr-Abl融合基因,以期应用于慢性粒细胞白血病(CMl)辅助诊断。方法采用重叠延伸法构建bCr-Abl融合基因阳性模板,设计并优化检测bCr-Abl融合基因的多重rT-PCr联合毛细管电泳体系,对检测体系进行初步的性能评估。结果多重rT-PCr技术检测bCr-Abl融合基因(E1A2、E13A2和E14A2)的最低检测限在102~103拷贝/μl;50例临床确诊CMl的患者应用该法检测bCr-Abl融合基因的阳性率为86.0%,其中E13A2型(20.0%),E14A2型(66.0%);本方法与骨髓细胞培养染色体核型分析相比,阳性符合率为97.6%,阴性符合率为75.0%,总符合率为94.0%,2种方法具有较高的一致性(kAPPA=0.765,P>0.05)。结论成功建立检测bCr-Abl融合基因的多重rTPCr联合毛细管电泳方法。该方法操作简单快捷、特异性好、灵敏度高,适合于临床上CMl的辅助诊断和分子分型。Objective To establish a method for detecting BCR-ABL fusion gene by multiplex reverse transcription polymerase chain reaction( RT-PCR),and to provide a useful tool for chronic myeloid leukemia( CML) auxiliary diagnosis.Methods The construction of BCR-ABL fusion gene as positive template was made by overlap extension method.The method of multiplex RT-PCR combining with capillary electrophoresis was designed and optimized.Its primary performance was evaluated.Results The lower detection limit for BCR-ABL fusion gene( ela2,e13a2 and e14a2) by multiplex RT-PCR was 102-103 copies / μL.The positive rate of this method to detect 50 CML patients was 86.0%( e13a2: 20.0% and e14a2: 66.0%).Compared with the results of chromosome karyotype analysis,the positive coincidence rate was 97.6%,and the negative coincidence rate was 75.0%.The 2 methods have high consistency( Kappa = 0.765,P > 0.05).Conclusions The method of multiplex RT-PCR combining with capillary electrophoresis to detect BCR-ABL fusion gene is simple and rapid with good specificity and sensitivity and is very suitable for the clinical detection of CML.福建省自然科学基金计划资助项目(2012J01414); 厦门市科技局计划项目资助课题(3502Z20113013

Efficacy and safety of raltegravir-based therapy in antiretroviral-naive patients with HIV-1 infection: A Meta-analysis

目的评价联合雷特格韦治疗方案用于人类免疫缺陷病毒-1 human immunodeficiency virus; 1,HIV-1)初次抗病毒治疗的安全性和有效性。方法检索联合雷特格韦用于HIV-1感染者初次抗病毒治疗的临床随机对照试验(randomized; controlled trial,RCT),采用Rev Man 5.2软件和Stata; 12.0进行Meta分析。结果纳入17个RCT,Meta分析结果:以血浆病毒载量0.05);治疗240周,试验组有效率高于对照组(70.2%和61.5%),合并效应量(RR = 1.15,95% CI: 1.03 ~; 1.28,P = 0.010)。试验组与对照组在腹泻、恶心、头晕、头痛、失眠等常见不良反应差异均无统计学意义(均有P; >0.05);其中96周及144/156周时脂质代谢异常增高合并效应量RR(95%; CI)分别低密度脂蛋白0.16(0.05~0.49)、0.20(0.08~0.48),甘油三酯0.12(0.02~; 0.59)、0.12(0.03~0.59),总胆固醇0.04(0.00~0.40)、0.04(0.00 ~; 0.34)。脂质代谢指标异常增高发生率,试验组均少于对照组,差异均有统计学意义(均有P 0.05) between the; raltegravir group and control group, proportion of patients with; virological response (plasma viral load 0.05) at weeks 48 and 96, respectively.; At weeks 96,144/156, the mean changes from baseline in LDL-cholesterol; (LDL) 0.16(0.05-0.49), 0.20 (0.08-0.48), triglyceride (TG); 0.12(0.02-0.59), 0.12(0.03-0.59) and total cholesterol (TC),; 0.04(0.00-0.40), 0.04(0.00-0.34) concen-trations, the raltegravir-based; regimens group had a lower incidence rate than the control group (all P; < 0.05). Conclusions Current evidence shows that the raltegravir-based; therapy had long-term and well tolerated antiretroviral activity, which; was non-inferior to current regimens recommended for; antiretroviral-native adults and adolescents with HIV-infection. Due to; limited quality and quantity of the included studies, more high quality; studies would be needed to verify the above conclusion.国家自然科学基金; 福建省科技创新平

Problems in China pharmaceutical compulsory licensing system and their solutions

面对全球公共健康的严峻形式,《多哈宣言》赋予WHO成员国处理公共健康危机时,具有使用药品专利强制许可的权利。虽然我国已基本建立药品专利强制许可制度,但是在实际工作中存在实施效力较低、行政主管部门职责不清、制药企业对有关规定了解不足等诸多问题,需要改进、完善有关法律规定和相关制度,切实提高药物可及性。The Doha Declaration has entrusted to the members of WHO the right to use the compulsory licensing for drug patents in the face of global public health crises. Although the basic compulsory licensing system for drug patents has been established in China,the efficiency of its implementation is low,the responsibility of its executive departments is unclear,the related rules are not fully understood by pharmaceutical enterprises,it is thus necessary to improve and perfect the relevant legal provisions and systems in order to effectively improve the accessibility of drugs

Preliminary analysis of newly diagnosed acute myeloid leukemia from 2005 to 2012 in Zhongshan Hospital Xiamen University

目的分析厦门大学附属中山医院近年来急性髓性白血病的发病情况与趋势,从中了解本地区急性髓性白血病的流行趋势,为制定科学的防治方法提供依据。方法回顾性分析2005~2012年厦门大学附属中山医院就诊的初发急性髓性白血病病例资料,统计分析年龄、性别、亚型及疗效的差异。结果新发急性髓性白血病患者的男女比例相当,无差异,25至59岁的中青年为高发病人群,其中35至39岁年龄段最高发,75到79岁出现老年人发病小高峰;M2、M3、M5是发病人数最多的三个亚型,且在未成年人、成年人、老年人不同年龄段组的发病比例有显著差异;2005~2012年新增患者人数具有上升趋势。结论 2005~2012年本院新发急性髓性白血病人数呈上升趋势,不同年龄段新发病例疗效差异显著可能与疾病亚型相关。Objective To explore the morbidity and trend of acute myeloid leukemia of Zhongshan Hospital Xiamen University in recent years, and to provide support for its prevention and intervention.Methods The retrospective analysis of acute myeloid leukemia patient of Zhongshan Hospital Xiamen University from 2005 to 2012 was made to analyze the differences among age, gender, subtype, and curative effect.Results The ratio of newly diagnosed acute myeloid leukemia between male and female was nearly same.The incidence of young and middle aged population between 25 to 59 years old was high.Among them, the highest incidence appeared in people aged 35 to 39.There was a small peak of incidence in old population aged 75 to 79.M2, M3, M5 were the most common subtypes of all patients, and there was a significant difference among age groups of minor, adult, and senior citizens.The newly diagnosed acute myeloid leukemia of each year was upward trend from 2005 to 2012.Conclusion The trend of newly diagnosed acute myeloid leukemia appeared upward.There was a significant difference of curative effects in newly diagnosed group of different ages may be caused by the specific subtype.福建省自然科学基金计划资助项目(2012J01414); 厦门市科技局计划项目资助课题(3502Z20113013); 福建省卫生厅青年课题(2013-2-92

人乳头瘤病毒18型病毒样颗粒在大肠杆菌中的表达及免疫原性分析

利用大肠杆菌表达系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,经过纯化和重组装过程获得HPV18病毒样颗粒(VLPs),研究其免疫原性和诱发中和抗体生成的水平。首先,提取HPV18的基因组DNA,通过PCR扩增获得HPV18 L1基因片段,将其插入pTrxFus表达载体,在大肠杆菌中可溶性表达HPV18 L1蛋白;其次,通过硫酸铵沉淀、离子交换层析和疏水相互作用层析获得高纯度的HPV18 L1蛋白,而后透析去除预先加入的还原剂DTT,使HPV18 L1蛋白自发组装成VLPs;最后,通过动态光散射技术和透射电子显微镜鉴定HPV18 VLPs的大小和形态,利用假病毒细胞中和实验评价HPV18 VLPs在实验动物体内的免疫原性和中和抗体生成水平。结果表明,HPV18L1蛋白可以在大肠杆菌表达系统中以可溶形式表达,经过纯化的HPV18 L1蛋白可以自发组装成为半径约为29.34nm、与HPV病毒外观相似的VLP。该VLPs在小鼠体内的中和抗体半数有效剂量为0.006μg,在兔及山羊体内诱导中和抗体滴度高达107。总之,本研究利用原核表达系统可简便高效地获得具有高度免疫原性的HPV18 VLPs,为HPV18预防性疫苗的开发奠定了基础,具有重要的应用意义

戊型肝炎病毒基因1型和基因4型中和表位区域分子差异研究

戊型肝炎病毒(HEV)根据易感宿主的差别可以分为两大类:一类只分离自人的H(Human)类,包括HEV-1和HEV-2;一类为人畜共患的Z(Zoonosis)类,包括HEV-3和HEV-4。本研究通过比较这两类HEV的ORF2aa368~606区段,发现存在4个类保守的差异位点,均位于HEV的主要中和表位区域aa459~606,分别是aa483、aa492、aa497和aa599;对这四个位点进行定点替换突变,以一组能够捕获HEV-1和/或HEV-4的单克隆抗体比较各种突变体的免疫反应性,结果表明仅aa497的差异造成了这两类HEV中和表位构象的部分差异,提示aa497及其相关的病毒表面结构差异在H类和Z类HEV宿主选择中可能扮演重要角色

抗戊型肝炎病毒单克隆抗体识别表位的初步研究

通过Western blot、体外捕获PCR、ELISA阻断实验及合成的多肽库等方法,对23株抗戊型肝炎病毒(HEV)单克隆抗体(单抗)识别HEV ORF2表位的作用进行系统研究。结果显示,7株线性单抗识别表位都位于ORF2 aa408~458之间,16株构象型单抗识别表位都定位于ORF2 aa459~606之间,大部分构象型单抗识别表位都在天然病毒表面。对这些单抗识别表位系统地了解将为HEV疫苗、诊断、病毒受体和病毒感染机制等方面的研究提供重要工具

水痘-带状疱疹病毒糖蛋白gE在昆虫细胞中的表达鉴定及其晶体培养

目的利用杆状病毒-昆虫表达系统建立纯化水痘带状疱疹病毒(VZV)包膜糖蛋白gE的方法,筛选gE蛋白的晶体培养条件,以期用于结构解析。方法将VZV糖蛋白gE基因序列克隆至杆状表达载体pAcgp67B载体中,利用High FiveTM昆虫细胞表达gE蛋白并进行TALON亲和层析纯化;利用WAVE生物波浪反应器建立含硒代甲硫氨酸的gE蛋白方法,以便在晶体结构解析中利用单波长或多波长异常散射进行相位解析;通过分子排阻色谱、分析超离,差示扫描量热法和酶联免疫吸附试验等分析gE蛋白的理化性质;利用结晶试剂盒对gE498和gE354蛋白的结晶条件进行初筛。结果获得的gE498和gE354蛋白纯度约为90%、产量为8～10 mg/L。理化分析显示两种gE蛋白在溶液中主要以均一稳定的单体形式存在,并呈现出良好的反应原性。在gE354晶体初筛中获得3个结晶条件:CrystalH3、PEGH12和IndexB10。结论建立了VZV糖蛋白gE表达和纯化方法,制备的蛋白纯度高,且具有反应原性,并筛选出CrystalH3、PEGH12和IndexB10这3个结晶条件,为gE糖蛋白的结构与功能研究及新型疫苗开发奠定了基础。国家自然科学基金项目(No.81871648);;\n新药创制专项项目(No.2018ZX09711003-005