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

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SYBR^TM 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。

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. SYBR^TM 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.

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