用于动物细胞种属鉴定及交叉污染检测的同工酶电泳技术改良

Improvement of isoenzyme electrophoresis technique for animal celllines identification and cross-contamination detection

目的改良现有的用于动物细胞种属鉴定和交叉污染检测的同工酶电泳技术。方法在传统的同工酶电泳基础上,将巴比妥缓冲液换成Tris-Gly缓冲液,将聚丙烯酰胺凝胶换成琼脂糖凝胶,以两种人源细胞(人宫颈癌细胞系HeLa、人肺癌细胞系A549)、两株小鼠来源细胞(小鼠成纤维细胞系L929、NIH/3T3)为样本进行体系优化,通过分析3种常用同工酶(葡萄糖-6-磷酸脱氢酶G6PD、苹果酸脱氢酶MD和核苷磷酸化酶NP)在优化的电泳条件下的酶谱,探索最适配方和电泳条件。结果改良后的15 mmol/L Tris-Gly琼脂糖凝胶同工酶电泳体系,能在室温条件下完成电泳及显色检测,获得的G6PD、MD、NP等3种酶的酶谱条带与传统的巴比妥缓冲体系一致,证明本体系优化成功。结论改良的Tris-Gly琼脂糖凝胶同工酶电泳能在室温操作、操作简易,可用于动物细胞种属鉴定及交叉污染的检测。

Objective To improve the existing isoenzyme electrophoresis technology for animal cell species identification and cross-contamination detection.Methods Based on traditional isoenzyme electrophoresis,the barbital buffer was replaced with Tris-Gly buffer,and the polyacrylamide gel was replaced with agarose gel.Two human cell lines[human cervical cancer cell line(HeLa),human lung cancer cell line(A549)]and two mouse cell lines(mouse fibroblast cell line,L929 and NIH/3T3)were used as samples for system optimization.The isoenzyme patterns of three commonly used enzymes[glucose-6-phosphate dehydrogenase(G6PD),malate dehydrogenase(MD),nucleotide phosphorylase(NP)]were analyzed under the optimized electrophoresis conditions to explore the most suitable formulation and electrophoresis conditions.The most suitable formulation and electrophoresis conditions were confirmed.Results The modified 15 mmol/L Tris-Gly agarose gel isoenzyme electrophoresis system was able to complete the electrophoresis and staining detection process at room temperature.The isoenzyme patterns obtained under this system were consistent with those under the traditional barbital buffer system,indicated the success of this system optimization.Conclusion The modified Tris-Gly agarose gel isoenzyme electrophoresis system could be operated at room temperature in brief and simple procedure.It can be used for animal cell species identification and cross-contamination detection.