Objective To study the biological activity of purified recombinant human epidermal growth factor ( rhEGF ) obtained from Samia cynthia ricini pupae bioreactor. Methods rhEGF was identify by Tricine-SDS-polyacrylamide gel electrophoresis( Tricine-SDS-PAGE ) and Western blot, then methyl thiazolyl tetrazolium( MTT ) assay and bypan blue staining technique were performed for the detection of the biological activity of balb/c3T3 cell. Results Successful expression of desired proteins could be observed by methods of Tricine-SDS-PAGE and Western blot. MTT assay showed that the purified rhEGF caused the promotion of the enzyme activity in Balb/c3T3 cell in a dose-dependent manner ranged from 0.0625 to 1.0 μg/L. The best concentration in enzyme activity promoting was 1.0 μg/L,and its effect was stronger than that of standard rhEGF( P <0.05 ). The purified rhEGF could stimulate the proliferation of Balb/c3T3 ,and its effect was much more stronger in proliferation stimulating compared with the standard rhEGP( P <0.05 ). Conclusion The purified rhEGF obtained from Samia cynthia ricini pupae bioreactor has similar biological activity with natural hEGF,which has a better effect compared with standard rhEGF. This research provides a basis for the further animal test of rhEGF.%目的 探讨蓖麻蚕蛹生物反应器中分离纯化获得的重组人表皮生长因子(rhEGF)蛋白质的生物学活性.方法 采用Tricine-SDS-PAGE和免疫印迹法(Western blot)鉴定rhEGF,通过MTT和台盼蓝法检测rhEGF的细胞活性.结果 Tricine-SDS-PAGE、Western blot结果显示,蓖麻蚕蛹中分离纯化获得的rhEGF具有正确的分子量和免疫原性;MTT法显示,纯化的rhEGF具有酶促活性,且酶促活性优于rhEGF标准蛋白(P＜0.05),其最高活性浓度为1.0 μg/L,在0.0625～1.0 μg/L浓度范围内具有剂量依赖性;rhEGF能够促进Balb/c3T3细胞的分裂增殖,效果优于rhEGF标准蛋白(P＜0.05).结论 AnpehEGF感染的蓖麻蚕蛹中分离纯化获得的rhEGF,具有类似天然hEGF的生物学活性,且效果优于rhEGF标准蛋白;rhEGF的细胞实验为进一步的动物实验提供了基础.
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