胶原三股螺旋重复蛋白1基因真核表达载体的构建及其与microRNA-30b关系初探

周辉芳; 隗和儒; 马士朝; 张超群; 王君平; 杨新英; 谢英; 孙殿兴

doi:10.3969/j.issn.1001-5256.2020.02.029

胶原三股螺旋重复蛋白1基因真核表达载体的构建及其与microRNA-30b关系初探

DOI: 10.3969/j.issn.1001-5256.2020.02.029

1. 河北医科大学研究生学院
2. 中国人民解放军联勤保障部队第九八〇医院全军肝病中心
3. 承德医学院
4. 河北医科大学实验动物学部河北省实验动物重点实验室

基金项目:

国家自然科学基金资助项目(30571667)；河北省科技支撑计划项目(15967777D)；

详细信息

中图分类号: R735.7
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出版历程
- 收稿日期: 2019-08-15
- 出版日期: 2020-02-20

Construction of the eukaryotic expression vector of collagen triple helix repeat containing 1 gene and its association with microRNA-30b

Graduate School of Hebei Medical University

Research funding:

摘要

摘要:
目的构建pCTHRC1-IRES2-EGFP真核表达载体,初步探讨胶原三股螺旋重复蛋白1基因（CTHRC1）与microRNA（简写为miR）-30b的关系。方法设计特异性引物,通过PCR扩增合成含有CTHRC1 CDS+3’UTR区的序列,并克隆至p IRES2-EGFP真核表达载体,得到p CTHRC1-IRES2-EGFP真核表达载体。采用NheⅠ/XhoⅠ双酶切电泳和测序法进行鉴定;通过脂质体转染法转染Huh7及HepG2细胞评价转染效率;将miR-30b mimic、control、pCTHRC1-IRES2-EGFP质粒和pIRES2-EGFP质粒转染至293T细胞中,分别采用Western Blot和实时荧光定量PCR法检测转染48 h后CTHRC1蛋白的表达水平及miR-30b的表达水平,初步探讨CTHRC1基因与miR-30b的关系。两组间比较采用独立样本t检验。结果采用NheⅠ/XhoⅠ双酶切pCTHRC1-IRES2-EGFP真核表达载体,得到5300 bp和1100 bp两条片段,与预期结果一致,测序结果也显示序列与GeneBank公布序列完全一致,表明p CT...
- 胶原三股螺旋重复蛋白1 /
- 质粒 /
- 细胞系,肿瘤 /
- 微RNAs
Abstract:
Objective To construct pCTHRC1-IRES2-EGFP eukaryotic expression vector,and to investigate the association between collagen triple helix repeat containing 1( CTHRC1) gene and miR-30 b. Methods Specific primers were designed and PCR was used for the amplification and synthesis of the sequence of CTHRC1 CDS + 3 'UTR region,which was then cloned into the pIRES2-EGFP eukaryotic expression vector to obtain the p CTHRC1-IRES2-EGFP eukaryotic expression vector. Nhe I/Xho I double enzyme electrophoresis and sequencing were used for identification,and the lipofection method was used to evaluate the transfection efficiency of Huh7 and HepG2 cells.After miR-30 b mimic,control,p CTHRC1-IRES2-EGFP plasmid,and pIRES2-EGFP plasmid were transfected into 293 T cells,Western Blot and quantitative real-time PCR were used to measure the protein expression of CTHRC1 and the expression of miR-30 b at 48 hours after transfection. The association between CTHRC1 gene and miR-30 b was analyzed. The independent samples t-test was used for comparison between two groups. Results Nhe I/Xho I double enzyme digestion was performed for the p CTHRC1-IRES2-EGFP eukaryotic expression vector,and two fragments with a length of 5300 bp and 1100 bp were obtained,which was consistent with the expected results;the sequencing results also showed that the sequence was exactly the same as that published in GeneBank,suggesting that the pCTHRC1-IRES2-EGFP eukaryotic expression vector was constructed successfully. In the transfection of Huh7 and HepG2 cells,2 μg p CTHRC1-IRES2-EGFP plasmid had a transfection efficiency of 90% and 89%,respectively,while 4 μg p CTHRC1-IRES2-EGFP plasmid had a transfection efficiency of 85% and 83%,respectively. In 293 T cells,the expression of CTHRC1 decreased to the miR-30 b group( P <0. 05). The expression of miR-30 b decreased in the group with overexpressed CTHRC1( P < 0. 05). Conclusion The eukaryotic expression vector of pCTHRC1-IRES2-EGFP is successfully constructed,and the expression of CTHRC1 is negatively correlated with the expression of miR-30 b in 293 T cells.
- collagen triple helix repeat protein 1 /
- plasmids /
- cell line,tumor /
- microRNAs

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