抑癌基因Fhit重组腺病毒的构建表达及其在结肠癌细胞中的生物学功能

中国生物工程杂志

2010, Vol. 30

Issue (01): 7-11

研究报告

抑癌基因Fhit重组腺病毒的构建表达及其在结肠癌细胞中的生物学功能

余桂戎^1,2,张瑞^1,2 ,孟艳玲^1,3 ,王磊^1,2 ,高飞⁴,郭旭⁵,赵晶^1,2 ,王涛^1,3,杨安钢^1,2,3**

1.第四军医大学国家肿瘤重点实验室
2.第四军医大学生物化学与分子生物学教研室
3.第四军医大学免疫学教研室
4.第四军医大学口腔医学系5第四军医大学细胞生物工程中心西安 710033

Construction and Expression of Recombinant Adenovirus Encoding Tumor Suppressor Fhit and Its Biological Function in Colon Cancer Cells

YU Gui-rong^1,2,ZHANG Rui^1,2,MENG Yan-ling^1,3,WANG Lei^1,2,GAO Fei⁴,
GUO Xu⁵,ZHAO jing^1,2,WANG Tao^1,3,YANG An-gang^1,2,3

1.State Key Laboratory of Cancer Biology
2.Department of Biochemistry and Molecular Biology
3.Department of Immunology
4.Department of Stomatology5 Cell Engineering Research Centre, Fourth Military Medical University, Xi’an 710033, China

全文: PDF(529 KB) HTML

摘要：

目的：构建携带抑癌基因Fhit的重组腺病毒并通过其研究Fhit蛋白对结直肠癌细胞增殖能力的影响。方法：利用PCR方法从人胎肝文库中克隆Fhit基因片段，Fhit基因PCR产物连入T载体构建重组质粒pMD18T-Fhit。测序正确后，将基因片段导入ptrack-CMV穿梭质粒中，构建重组穿梭载体ptrack-CMV-Fhit。将经PmeⅠ 单酶切线性化的ptrack-CMV-Fhit和骨架腺病毒质粒pAdEasy-1共转BJ5183大肠杆菌，使ptrack-CMV-Fhit和pAdEasy-1发生同源重组。经PacI酶切鉴定正确后,将重组腺病毒质粒转染293A细胞获得表达Fhit蛋白的重组腺病毒rAd-Fhit，将获得的重组腺病毒感染结肠癌细胞，采用蛋白印迹法检测外源Fhit蛋白的表达，并进一步观察其对细胞增殖能力的影响。结果：我们成功构建了携带有Fhit基因的重组腺病毒，且能够在结肠癌细胞中成功表达Fhit蛋白。在结肠癌细胞中，Fhit蛋白明显减弱了结肠癌细胞的增殖能力。结论：在结肠癌细胞中，Fhit基因可能扮演抑癌基因的角色，而表达Fhit的重组腺病毒很可能成为一种结肠癌生物治疗的新策略。

关键词： ; Fhit; 重组腺病毒; 结肠癌; 细胞增殖

Abstract:

AIM: To construct a recombinant adenovirus carrying Fhit gene, a tumor suppressor in many types of cancer, and to observe its biological function on the proliferation of colon cancer cells. METHODS: Fhit gene was cloned from the fetal liver cDNA library using the PCR method. The PCR product was inserted into the T vector to construct the plasmid pMD18T-Fhit. The Fhit fragment from the pMD18T-Fhit was inserted into the vector ptrack-CMV to construct a shuttle plasmid ptrack-CMV-Fhit. After PmeI digested and linearized process, ptrack-CMV-Fhit was co-transformed into Escherichia coli strain BJ5183 together with the adenovirus backbone vector pAdEasy-1 to generate a recombinant adenovirus plasmid by homologous recombination. The adenovirus plasmid was identified by PacI digestion and transfected into 293A cells to package a recombinant adenovirus which expressed the Fhit protein. Furthermore, the adenovirus rAd-Fhit was infected into colon cancer cells，and the expression of the ectogenic protein was detected by Western blotting. Finally, the proliferation of colon cancer cells was observed in adenovirus-infected cells by the MTT assay. RESULTS: We constructed the recombinant adenovirus encoding Fhit gene and expressed it in colon cancer cells successfully. We detected that the proliferation of colon cancer cells was inhibited obviously in rAd-Fhit-infected cells with comparison to the control groups. CONCLUSION: Fhit may function as a tumor suppressor in colon cancer cells, and the adenovirus-mediated Fhit can be a novel strategy for the colon cancer therapeutics.

Key words: Fhit adenovirus colon cancer proliferation

收稿日期: 2009-10-29 出版日期: 2010-01-27

基金资助:

国家自然科学基金（30873006，30901771）、第四军医大学“青年英才支持计划”（2009）、第四军医大学学员课外科研课题（2008）资助项目

通讯作者: 杨安钢 E-mail: agyang@fmmu.edu.cn

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引用本文:

余桂戎张瑞孟艳玲王磊高飞郭旭赵晶王涛杨安钢. 抑癌基因Fhit重组腺病毒的构建表达及其在结肠癌细胞中的生物学功能[J]. 中国生物工程杂志, 2010, 30(01): 7-11.

TU Gui-Reng, ZHANG Rui, MENG Yan-Ling, WANG Lei, GAO Fei, GUO Xu, DIAO Jing, WANG Chao, YANG An-Gang. Construction and Expression of Recombinant Adenovirus Encoding Tumor Suppressor Fhit and Its Biological Function in Colon Cancer Cells. China Biotechnology, 2010, 30(01): 7-11.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I01/7

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