摘要
目的构建内质网滞留型的抗CyclinD1胞内抗体并鉴定其在肿瘤细胞中的表达活性。方法利用PCR技术将细胞内质网滞留信号及E-tag检测标签引入抗CyclinD1人源单链抗体,获得抗CyclinD1胞内抗体(AD5E)基因片段,通过分子克隆技术,构建重组真核表达载体pcDNA3.1-AD5E,限制性内切酶分析及DNA测序验证其序列及读码框正确后,利用脂质体介导法转染HeLa细胞,RT-PCR和间接免疫荧光实验分析该胞内抗体在肿瘤细胞中的表达活性。结果限制性内切酶分析及DNA测序发现,正确引入细胞内质网滞留信号和E-tag检测标签序列,该胞内抗体片段为876bp。RT-PCR及免疫荧光实验结果显示,仅在转染胞内抗体基因的HeLa细胞中存在该胞内抗体的表达,并且主要分布在细胞质区域。结论我们成功构建了在肿瘤细胞中能够有效表达的内质网滞留型的抗CyclinD1胞内抗体。
Objective To construct endoplasmic reticulum-retained anti-cyclin D1 intrabody and then identify its expression in HeLa cells.Methods Using the anti-cyclin D1 scFv as template,endoplasmic reticulum-retained signal peptides （KDEL） and E-tag was introduced into anfi-cyclin D1 intrabody gene （ADSE） by PCR. Then the intrabody gene （ADSE） was subcloned into pcDNA3.1 to construct the recombinant expression vector pcDNA3.1-ADSE. Analysis of restriction endonuclease and DNA sequencing was con- ducted. The intrabody gene was transfected into human HeLa cells. RT-PCR and immunofluorescence was performed to detect the expression of this intrabedy. Results Analysis of restriction endonuclease and DNA sequencing showed that the intrabody gene and expression vector were constructed successfully. The intrabody gene was 876 bp in length. RT-PCR and immunofluorescence showed that the mRNA and proteins were only detected in the HeLa cells transfected with intrabody and the intrabody was distributed mainly in the cytoplasm. Conclusion The anti-cyclin D1 intrabody with KDEL was developed successfully. This intrabody was found to be expressed efficiently in the cytoplasm of HeLa cells.
出处
《中国实验诊断学》
2008年第7期821-824,共4页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金青年基金资助项目(30200256)
吉林省科技发展计划资助项目(20050410-3)
吉林吉林省杰出青年基金(200600107)
