hTERT原核重组质粒的表达

目的本课题拟观察原核重组质粒pGEX4T1hTERT在BL21细菌中的表达,为进一步探讨以hTERT为靶点的肿瘤基因免疫治疗提供实验依据。方法IPTG诱导带有pGEX4T1hTERT质粒的BL21细菌融合蛋白的表达,取不同时间的培养物进行SDSPAGE分析,并进行薄层凝胶光密度扫描测定目的蛋白表达量,同时对表达产物进行Westernblot检测。结果对SDSPAGE结果分析发现,含pGEX4T1hTERT质粒的BL21细菌能够表达出63kD的融合蛋白。薄层凝胶光密度扫描测定其表达水平最高为28.4%,以IPTG诱导4h表达量最高。Westernblot检测表明该融合蛋白可被hTERT多抗识别。结论诱导pGEX4T1hTERT重组质粒,获得分子量为63kD的GSThTERT融合蛋白。用抗hTERT目的基因片段表达的相应抗原蛋白。

Expression of hTERT Prokaryotic Recombinant Plasmids

Objective To provide a sound basis for further researching cancer immunogene therapy, and to observe the expression of pGEX-4T-1-hTERT in BL21.Methods BL21 containing pGEX-4T-1-hTERT was inducted by 1 mmol/L IPTG, and then was analyzed on SDS-PAGE of 12% gel. The fusion protein was also ananlyzed by Western-blot.Result A 63 kD protein band of predicted molecular mass which presumably comprised a GST and an hTERT was observed with samples induced by IPTG,but not in the samples without IPTG induction. The level of expression peaked at 4h post-incubation. The portion of the fusion protein reached 28.4% of all the proteins by thin-layer gel optical scanning. The fusion protein was also analyzed by Western-blot. The results showed that the fusion protein had the better antigenicity and could be recognized by hTERT polyclone antibody.Conclusion Western blot analysis confirmed that 63 kD fusion protein of pGEX-4T-1-hTERT could be specifically recognized by hTERT polyclone antibody. This result suggested that there was antigenic protein of hTERT gene encoding.