关闭
 
读者在线:用户名 密码
首页 期刊简介 投稿须知 期刊目录 专家风采 编委会 特邀顾问 联系我们 移动出版
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5



刊物信息

期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

访问统计
您是第  4 8 5 1 4 0 0 位浏览者
您当前的位置:首页 >> 正文

应用CE-LIF方法分析重组人1型单纯疱疹病毒的DNA限制酶酶切片段

Analysis of DNA restriction enzyme fragments of recombinant human herpes simplex virus type 1 by capillary electrophoresis with laser-induced fluorescence detection

分类号:R917
出版年·卷·期(页码):2020,40 (1):37-42
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的: 建立毛细管电泳-激光诱导荧光(CE-LIF)检测方法,对重组人1型单纯疱疹病毒(rHSV-1)的DNA限制性酶切片段进行分析。方法: 以rHSV-1为研究对象,用核酸提取仪提取其DNA,以DNA限制性内切酶Nde Ⅰ酶切,以SYBRTM Gold nucleic acid作为荧光标记物,利用CE-LIF实现分离检测,通过DNA ladder计算片段大小。CE-LIF条件:采用涂层毛细管(有效长度30 cm,总长度40 cm,内径100 μm),3.45 kPa压力进样3 s;毛细管分离温度设为25℃,毛细管分离电压为-7 kV,分离时间40 min;LIF检测器的激发波长及发射波长分别为488 nm和520 nm。结果: 新建方法可以对8个目标酶切片段分离检测,各片段峰迁移时间的RSD(n=6)均小于0.35%,片段峰1~7校正峰面积百分比的RSD(n=6)均小于5.6%,方法精密度良好。通过DNA ladder回归方程,对DNA酶切产物进行片段大小的计算,各片段大小计算结果RSD(n=6)均小于5.8%,且与琼脂糖凝胶电泳法测得结果相一致。结论: 本研究建立的rHSV-1的DNA限制酶酶切片段CE-LIF分析法自动化程度高,灵敏度高,重复性好,试剂消耗及环境污染小,适用于rHSV-1的鉴别分析。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To analyze the DNA restriction fragments of recombinant human herpes simplex virus type 1(rHSV-1) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Methods: The DNA of rHSV-1 was extracted by nucleic acid extractor and digested by DNA restriction endonuclease Nde Ⅰ. SYBRTM gold nuclear acid was used as fluorescent dye. The labeled DNA fragments were separated and detected by CE-LIF and their sizes were calculated based on DNA ladder. The CE-LIF was operated with a coated fused silica capillary tube (effective length:30 cm; total length:40 cm; internal diameter:100 μm) under 25℃,-7 kV for 40 min. The sample was injected under 3.45 kPa for 3 s. The excitation wavelength and emission wavelength were 488 nm and 520 nm separately. Results: This method could separate and detect eight target fragments. RSD of the peak migration time of each fragment was less than 0.35%(n=6), and the RSD of the corrected peak area percentage of fragment peak 1-7 was less than 5.6%(n=6), indicating good precision of the method. The sizes of DNA fragments were calculated by regression equation of DNA ladder. For each fragment, RSD was less than 5.8%(n=6), and the calculated size was consistent with the result of agarose electrophoresis. Conclusion: The CE-LIF method established in this study has the advantages of high automation, high sensitivity, good repeatability, low reagent consumption and environmental pollution, which is suitable for the identification and analysis of rHSV-1 vector of gene therapy drugs.

-----参考文献:---------------------------------------------------------------------------------------

[1] SLOBODKINA EA, MAKAREVICH PI, DOLINKIN AO.Issues of the development of gene therapy drugs[J].Russ J Biopharm, 2018, 10(5):3
[2] AFIONE SA, CONRAD, CK, FLOTTE TR.Gene therapy vectors as drug delivery systems[J].Clin Pharmacokinet, 1995, 28(3):181
[3] BOUARD D, ALAZARD-DANY D, COSSET FL.Viral vectors:from virology to transgene expression[J].Br J Pharmacol, 2009, 157(2):153
[4] HARSTAD RK, JOHNSON AC, WEISENBERGER MM, et al.Capillary electrophoresis[J].Anal Chem, 2016, 88(1):299
[5] LI LS, ZHAO Y, WANG HM, et al.Annual review of capillary electrophoresis technology in 2018[J].Chin J Chromatogr, 2019, 87(5):463
[6] ATTALLAH AM, ABDALLAH SO, EL-DESOUKY MA, et al.A rapid, low-cost quantitative diagnostic method for hepatitis C virus infection using capillary zone electrophoresis[J].Eur J Clin Microbiol Infect Dis, 2014, 33(3):439
[7] BELOV AM, VINER R, SANTOS MR, et al.Analysis of proteins, protein complexes, and organellar proteomes using sheathless capillary zone electrophoresis-native mass spectrometry[J].J Am Soc Mass Spectrom, 2017, 28(12):2614
[8] KOSTAL V, KATZENMEYER J, ARRIAGA EA.Capillary electrophoresis in bioanalysis[J].Anal Chem, 2008, 80(12):4533
[9] SUN H, LI L, SU M.Simultaneous determination of proline and pipemidic acid in human urine by capillary electrophoresis with electrochemilum-inescence detection[J].Clin Lab Anal, 2010, 24(5):327
[10] 中华人民共和国药典2015年版.三部[S].2015:通则29 ChP 2015.Vol Ⅲ[S].2015:General rule 29
[11] MANSERVIGI R, ARGNANI R, MARCONI P.HSV recombinant vectors for gene therapy[J].Open Virol J, 2010,4:123
[12] WORKENHE ST, SIMMONS G, POL JG, et al.Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes totherapeutic efficacy[J].Mol Ther, 2014, 22(1):123
[13] SLATER GW.DNA gel electrophoresis:the reptation model(s)[J].Electrophoresis, 2009, 30(Suppl 1):S181

欢迎阅读《药物分析杂志》!您是该文第 343位读者!

药物分析杂志 © 2009
地址:北京天坛西里2号 邮政编码:100050; 电子邮件:ywfx@nicpbp.org.cn

本系统由北京博思汇文数字科技有限公司设计开发 技术服务电话:400-921-9838