Flow-injection-enhanced chemiluminescence method for the determination of three β-blockers

Objective To develop a rapid, simple and sensitive chemiluminescence method for the determination of three β-blockers (bisoprolol, atenolol and propranolol). Methods The chemiluminescence of cerium (Ⅳ)-sulfite system was obviously sensitized by adding anyone of three β-blockers in acid media. A new chemiluminescence method was set up by combining with flow-injection technique and used to determine the three β-blockers. Results Good linear ranges were obtained at the concentrations of 2.0×10-7g/mL-4.0×10-5g/mL, 1.0×10-7g/mL-3.0×10-5g/mL and 7.0×10-7g/mL-1.0×10-5g/mL, respectively, with the detection limits of 5.0×10-8g/mL, 7.0×10-8g/mL and 5.0×10-8g/mL (S/N=3), respectively, and the relative standard deviations for 11 times consecutive injections of 1.0×10-6g/mL bisoprolol, atenolol and propranolol were 3.57%, 2.21% and 2.26%, respectively. Conclusion The developed method is sensitive, accurate, rapid and of low cost. And it can be applied to determine bisoprolol, atenolol and propranolol in pharmaceutical preparations.     

RP-HPLC method for the quantitation of β-Sitosterol in Elaeagnus Gon yanthes Benth

Objective To establish an RP-HPLC method for the determination of β-Sitosterol in Elaeagnus Gonyanthes Benth. Methods The separation was performed on a luna C8 (2) (150 mm×4.6 mm, 5μm) column with the mobile phase of methanol-water (88∶12, v/v) at a flow rate of 1.0 mL/min, the detection wavelength was set at 210 nm, and the temperature of the column was maintained at 35 ℃. Results The calibration curve of β-Sitosterol was linear over the concentration range of 0.075-0.375 mg/mL (r=0.9999) and the average recovery of β-Sitosterol was 96.30% with RSD of 3.60%(n=3). Conclusion The method is simple, rapid, and accurate, and can be used for the quality control of Elaeagnus Gonyanthes Benth.     

DETERMINATION OF TETRACYCLINE AND OXYTETRACYCLINE BY FLOW-INJECTION CHEMILUMINESCENCE METHOD

Tetracycline and oxytetracycline are broad-spec-trumantibiotics.They are not only used in humanpathologies,but alsoin veterinary medicine,ani malnutrition and feed additives for cattle breeding.In the past few years,the deter mination meth-ods of tetracycline and oxytetracycline were repor-ted,which involved in difference spectrophotome-try[1],HPLC[2,3],spectrophotometry[4-7],HPLC-MS[8-9],spectrofluori metry[10],solid-phase extrac-tion[11]and kinetic methods.Recently,flow-injection CL met…     

Capillary electrophoresis coupled with electrochemiluminescence for determination of cloperastine hydrochloride

Objective To investigate the electrochemiluminescence (ECL) behavior of cloperustine hydrochloride.Methods ECL intensity of tris (2,2'-bipyridyl) rutheniumo(Ⅱ) was enhanced, the method for the determination of dupernstine hydrochloride was established using capillary electrophoresis (CE) coupled with electrochemilumolinescence (ECL) detection. Results Under the optimum conditions, ECL intensity varied linearly with cloperastine hydrochloride concentration from 7.0×10-6 g/mL to 1.0×10-4 g/mL. The detection limit (S/N=3) was 8.05×10-7g/mL. he relative standard deviation of the ECL intensity and the migration time for 11 consecutive injections of 1.0 ×10-5g/mL cloperastine hydrochloride was 2.9% and 1.5%, respectively. This method was successfully applied to eloperastine hydrochloride tablet determination. Conclusion The method has been established, validated and applied for determination of cloperastine hydrochloride.     

Analysis of ganciclovir and its related substances using high performance liquid chromatography and liquid chromatography-mass spectrometry methods

Objective High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) methods were developed for the determination of ganciclovir and its related substances. Methods A Hypersil ODS2 column (4.6mm×250mm, 5μm) was used with a mobile phase of 0.02M potassium 1.0mL/min, and UV detector set at 254nm was used for monitoring the eluents. Results The method was simple, rapid, selective and capable of separating all related substances at trace level with a detection limit of 0.04μg/mL. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 10.2-153.0μg/mL with r=0.9998. The percentage recoveries ranged from 96.7% to 101.6%, and RSD was 1.24%-1.96% (n=5). Conclusion The method was found to be suitable not only for monitoring the reactions during the process development but also for quality control of ganciclovir. For identification of related substances, LC/MS was used. The mainly related substances of ganciclovir active pharmaceutical ingredients (API) were determined as guanine, (1, 3-dioxolan-4-yl) methyl acetate, and diacetyl guanine.     

人神经生长因子成熟蛋白基因片段在大肠杆菌中的表达和生物活性鉴定

目的　在大肠杆菌中表达人神经生长因子成熟蛋白基因片段(hNGFβ)并检测其生物活性。方法　将 NGFβ目的基因亚克隆入表达载体pBV220的BamHI- BamHI位点,连接产物转化大肠杆菌DH5α菌株通过热诱导表达NGFβ蛋白,利用溶解包涵体和重折叠使表达产物复性,将复性后的蛋白质加入体外培养的鸡胚背根神经节及 PC12 细胞BrdU掺入实验,观察表达蛋白的生物学活性。结果　成功构建了重组质粒 pBV220/NGFβ。使用该质粒转化 DH5α宿主菌,使用热诱导方法表达了NGFβ蛋白,SDS PAGE结果提示表达蛋白主要存在于包涵体中。通过肝素亲和层析可以获得纯度大于90%的 NGFβ。每升表达菌可以获得 1.8~2.0 mg NGFβ。鸡胚背根神经节突起生长实验和PC12细胞培养生长刺激BrdU掺入实验表明原核表达的 NGFβ是具有生物活性的。它的生物活性按生物制品鉴定章程判断为1×105 BU/g。结论　在大肠杆菌中可以表达出有活性的NGFβ蛋白。     

Luminol-potassium permanganate chemiluminescence system for the determination of three anthracycline antibiotics

Objective To establish a flow-injection chemiluminescence method for the determination of doxorubicin, epirubicin and mitoxantrone and study its reaction mechanism. Methods In alkaline medium, chemiluminescence of luminol-potassium permanganate system could be inhibited obviously by anthracycline antibiotics. Combined with flow-injection technique, a new chemiluminescence method for determining the anthracycline antibiotics was set up. The chemiluminescence mechanism of the luminol-potassium permanganate system was also discussed. Results Under optimal conditions, the good linear ranges of doxorubicin, epirubicin and mitoxantrone were 5.0×10-9-1.0×10-6g/mL, 1.0×10-9-1.0×10-5g/mL and 3×10-9-1.0×10-6g/mL, respectively. The detection limits of doxorubicin, epirubicin and mitoxantrone were 3.0×10-9g/mL, 5.0×10-8g/mL and 2.0×10-9g/mL, respectively. During eleven repeated inter-day and intra-day precision tests of 1.0×10-6g/mL samples, the relative standard deviations corresponded to reference values of 3.0%, 2.8% and 2.1%. Conclusion The developed method is sensitive, accurate, rapid and of low cost. It can be applied to determine doxorubicin hydrochloride, epirubicin hydrochloride and mitoxantrone hydrochloride in injection preparations.     

Analysis and determination of diterpenoids in unprocessed and processed Euphorbia lathyris seeds by HPLC-ESI-MS

Euphorbia lathyris （Caper spurge） is a toxic and potent Chinese materia medica （T/PCMM）. This study sought a method for identifying five diterpenoids （Euphorbia factors LI-L3, L7a, and Ls） with the spectra of UV and mass, quantifying three diterpenoids L1, L2, and L8 in crude extracts of unprocessed and processed E. lathyris seeds by liquid chromatography/ electrospray ionization mass spectrometry （LC-ESI-MS）. The analysis was achieved on an Agilent Eclipse XDB-C18 column （4.6 mm× 150mm i.d., 5 μm） with an isocratic elution with a mobile phase consisting of water and acetonitrile at a flow rate of 0.25 mL/min at column temperature of 30 ℃ and UV detection was set at 272 nm. An ESI source was used with a positive ionization mode. The calibration curve was linear in the ranges of 9.9-79 μg/mL for Euphorbia factor Lb 3.8-30.5μg/mL for Euphorbia factor L2, and 1.0-20.6 μg/mL for Euphorbia factor LB. The average recoveries （n=6） of three diterpenoids were 98.39%, 91.10% and 96.94%, respectively, with RSD of 2.5%, 2.4% and 2.1%, respectively. The contents of the three diterpenoids in processed E. lathyris seeds were 3.435, 1.367 and 0.286 mg/g, respectively, which decreased more sharply than those in unprocessed E. lathyris seeds which were 4.915, 1.944 and 0.425 mg/g, respectively. The method is simple, accurate, reliable and reproducible, and it can be applied to control the quality of unprocessed and processed E. lathyris seeds.     

Expression and Application of the Recombinant Non-Structural Proteins of FMDV

Foot-and-mouth disease virus (FMDV) is acausative agent of highly infectious and economi-cally important disease in cloven-hoofed animals.In Asian and South American countries, commer-cial vaccines of FMDV are widely used for control-ling the disease, but animals in North America andEurope are not inoculated with FMD vaccines.Identifying animals that have been infected withFMDV is important because infected cattle andsheep can become carriers of the virus[1,2]Althoughonly African buff…     

人B7.2(CD86)胞外区原核表达及其工程菌发酵培养的实验研究

目的　利用原核系统表达人B7.2 (IgV +C)并对工程菌发酵培养条件进行优化。方法　用聚合酶链反应 (PCR)技术从B7.2cDNA全长中克隆B7.2 (IgV +C) ,将PCR产物克隆入表达载体 pGEX 4T 3从而得到重组体 pGEX 4T 3 /hB7.2 (IgV +C) ,SDS PAGE及Westernblot用于检测目的蛋白的表达 ,同时对目的蛋白诱导表达时间及诱导剂浓度进行优化 ,对重组质粒的遗传稳定性进行鉴定。结果　Westernblot结果显示相应分子质量 5 5kD处有hB7.2 (IgV+C)与GST融合蛋白的高效表达 ,表达量占菌体总蛋白的 3 0 %左右。对工程菌进行发酵培养研究的结果表明 ,所构建的重组质粒在工程菌DH5α中传代稳定 ,未见质粒丢失 ,目的蛋白的表达不受影响 ,且在 3 7℃诱导 5h、IPTG终浓度为 4mmol·L- 1 时其表达量最多。结论　证明了利用原核系统表达人B7.2 (IgV +C)的可行性。     

亚洲牛带绦虫成虫延伸因子-1基因的表达、纯化及免疫学分析

目的 对亚洲牛带绦虫成虫延伸因子-1(elongation factor 1,EF-1)基因进行克隆、表达和免疫学初步研究.方法 将亚洲牛带绦虫成虫EF-1克隆到原核表达质粒pET-28a( )中,在大肠杆菌BL-21/DE3中用异丙硫代-β-D半乳糖苷诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,用蛋白印迹法(Western blotting)进行免疫学分析.结果 PCR、双酶切及DNA测序结果 均表明重组质粒pET-28a( )-EF-1构建成功.重组蛋白可被感染了亚洲牛带绦虫患者血清和猪血清识别,表明其具有免疫反应性.结论 亚洲牛带绦虫成虫EF-1基因可在原核表达系统中获得具有免疫学活性的表达,为进一步研究该蛋白的功能奠定了基础.     

结核杆菌DnaA蛋白的原核表达及免疫血清的制备和检测

目的 在大肠杆菌中表达重组结核杆菌DnaA蛋白,对表达产物进行纯化、鉴定后制备DnaA蛋白特异性免疫血清并进行Western blot检测.方法 以结核分枝杆菌标准菌株H37Rv基因组为模板,PCR扩增DnaA基因,构建DnaA原核表达质粒pET-DnaA,转化表达宿主大肠杆菌BL21(DE3),IPTG诱导重组蛋白表...     

丙型肝炎病毒NS3-NS4基因的表达及鉴定

目的　构建丙型肝炎病毒 (HCV)非结构蛋白NS3 NS4全长基因重组质粒并诱导表达 ,鉴定NS3 NS4蛋白的抗原性。方法　应用PCR技术从pUC19/HCV中扩增目的基因 ,构建重组表达质粒 ,进行原核表达 ,用SDS PAGE、ELISA技术对表达产物的抗原性进行鉴定。结果　成功构建、表达了重组质粒pBV2 2 0 /NS3 NS4 ,用 5 0份质控标准血清对表达蛋白质进行抗原性检测 ,与第二代诊断试剂相比 ,总符合率为 96 %。结论　全长NS3 NS4蛋白是一个优势免疫原性区 ,应该是HCVEIA诊断试剂的有效成分。     

构建及鉴定携带角蛋白启动子和人乳头瘤病毒16E6/E7基因的重组腺病毒

目的利用AdEasy系统构建携带人角蛋白启动子的人乳头瘤病毒-16(HPV-16)E6/E7基因重组腺病毒,并通过RT-PCR方法检测E6/E7基因的表达。方法应用PCR方法从含有HPV-16全基因序列的质粒上扩增E6/E7基因,构建pCDNA3.1(-)-K14-E6/E7-polA载体,扩增、酶切获得K14-E6/E7-polA片段插入腺病毒穿梭载体质粒pAdTrack上,构建重组穿梭载体pAdTrack-K14-E6/E7-polA,线性化后与骨架载体AdEasy-1在细菌BJ5183内同源重组得到腺病毒质粒pAd-K14-E6/E7-polA,经人胚肾293细胞包装后得到重组腺病毒pAd-K14-E6/E7-polA。氢化铯(CsCl)梯度离心纯化病毒,提取病毒再感染后的293细胞总RNA,通过RT-PCR方法检测E6/E7基因的表达。结果通过同源重组的方法构建了腺病毒pAd-K14-E6/E7-polA载体,经酶切和测序鉴定该质粒构建成功。293细胞包装3d后观察到绿色荧光蛋白(GFP)表达,CsCl梯度离心纯化最终获得7.2×1010pfu/mL滴度的重组病毒;用该滴度病毒重新感染293细胞3d后,提取细胞总RNA,RT-PCR检测E6/E7有表达。结论利用新型腺病毒载体AdEasy系统可在短期内制备同时表达GFP和E6/E7的重组腺病毒pAd-K14-E6/E7-polA。这将为进一步研究HPV-16E6/E7基因功能及利用基因治疗女性宫颈癌奠定了基础。     

反转录病毒载体介导的EGFP基因在SK-N-SH神经母细胞瘤细胞中的表达

目的构建携带报告基因增强型绿色荧光蛋白(EGFP)的反转录病毒载体,并且探讨病毒载体对SK-N-SH神经母细胞瘤细胞株的感染效率。方法使用基因重组技术构建携带EGFP基因的反转录病毒载体(RV/EGFP)。将稀释的RV病毒液感染NIH3T3细胞,计数NIH3T3荧光表达细胞的数量来确定病毒的浓度;使用RV载体感染SK-N-SH细胞,通过流式细胞学荧光检测明确RV在SK-N-SH细胞的感染效率。结果成功构建了基因转移载体RV/EGFP,确定重组病毒的浓度为8.3×106病毒颗粒/mL。在SK-N-SH细胞RV/EGFP稳定转染并有效表达。在SK-N-SH细胞EGFP的荧光表达显示RV的转移并且表达的效率达到30%以上。结论SK-N-SH细胞能被RV病毒载体有效感染,在转基因细胞株外源基因的表达长期稳定并且显示RV病毒载体具有良好的基因转移效率。     

Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coil BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyelonai antibody, with which we studied the function of NSSATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight: 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.     

新型EGFP标记的杆状病毒转移载体的构建及EGFP的制备

目的构建可表达增强型绿色荧光蛋白(EGFP)融合蛋白的昆虫杆状病毒转移载体,利用Sf-9细胞表达、制备EGFP。方法用PCR法从pEGFP质粒中扩增EGFP基因,NcoⅠ/SalⅠ双酶切,克隆入杆状病毒转移载体NcoⅠ/SalⅠ酶切位点之间,获得pFB-EGFP质粒;转化E.coliDH10Bac,通过转座子Tn7的介导,获得重组杆状病毒Ac-EGFP;感染Sf-9昆虫细胞;利用激光共聚焦显微镜和SDS-PAGE观察并检测EGFP的表达。非变性法纯化EGFP。结果荧光显微镜和激光共聚焦显微镜证实感染的Sf-9细胞可表达EGFP;通过SDS-PAGE获得的EGFP,大小为27ku。结论重组pFB-EGFP载体具备表达EGFP的能力,为分子生物学研究提供了一种新型可表达EGFP融合蛋白的昆虫杆状病毒表达载体。     

Determination of enantiomeric impurity of etomidate by high performance liquid chromatography

Objective To determine enantiomeric impurity of etomidate using high performance liquid chromatography. Methods (R)-etomidate and (S)-etomidate were isopropanol-n-hexane. The flow rate of the mobile phase was 0.5mL/min. The detected wavelength was 242nm. Results (R)-etomidate and (S)-etomidate could be separated completely under these conditions. The precision of (R)-etomidate was 1.57% (n=3). The limit of detection of (R)-etomidate was 4.25ng/mL. The average percentage content of (S)-etomidate was 0.09% in the samples. Conclusion The method was repeatable and sufficiently sensitive to determine the enantiomeric impurity of etomidate. It allows the quantitation of the impurities at the 0.085% (w/w) level relative to etomidate at a concentration of the test solution of 5mg/mL.     

奋乃静药物的流动注射化学发光测定方法的建立

目的建立快速测定奋乃静的流动注射化学发光新方法。方法在硝酸介质中,奋乃静能被硫酸铈氧化生成发光物质奋乃静砜,从而产生化学发光。基于此,建立了奋乃静的流动注射化学发光分析方法。结果在优化的实验条件下,不用任何发光增敏剂,奋乃静在1.0×10-7-7.0×10-5g/mL范围内与化学发光强度呈良好的线性关系,检出限(3σ)为8.0×10-8g/mL,对1.0×10-6g/mL的奋乃静进行了11次平行测定,其相对标准偏差为1.8%。结论本方法简便、快速、准确、灵敏度高、线形范围宽,应用于奋乃静片剂分析,并与药典方法进行对照,结果满意。     

Detection of captopril based on its enhanced resonance light scattering signals of fluorosurfactant-capped gold nanoparticles

In this study, based on its enhancement effect on resonance light scattering (RLS) of fluorosurfactant (FSN)-capped gold nanoparticles (GNPs), we reported a simple approach for the rapid sensing of captopril. Under optimum conditions, the lowest detectable concentration of captopril through this approach (S/N=3) was 0.01μg/mL. The calibration curve was linear over the range of 0.08-4.0μg/mL for the detection of captopril. The recoveries of captopril were found to fall in the range between 99% and 100%. We have validated the applicability of our method through the analyses of captopril in pharmaceutical formulations. Good agreements were obtained for the determination of captopril between the present approach and official method.