携带c-Met短发夹结构重组腺相关病毒的包装及滴度测定

doi:国家自然科学基金资助课题（30671173）

• 基础研究 • 下一篇

携带c-Met短发夹结构重组腺相关病毒的包装及滴度测定

赵慧¹，路英丽²，王凯忠³，王忠山²

(1.吉林大学基础医学院组织学与胚胎学教研室，吉林　长春　130021；2.吉林大学第二医院妇产科, 吉林　长春　130041；3.吉林大学第一医院胸外科，吉林　长春　130021）

收稿日期:2006-11-06 修回日期:1900-01-01 出版日期:2007-11-28 发布日期:2007-11-28
通讯作者: 王忠山

Package and titer assay of recombination adeno-associated viruswith c-Met short hairpin

ZHAO Hui¹,LU Ying-li²,WANG Kai-zhong³，WANG Zhong-shan²

(1.Department of Histology and Embryology,School of Basic Medical Sciences,Jilin University,Changchun 130021,China；2.Department of Obstetrics and Gynecology,Second Hospital,Jilin University,Changchun 130041,China；　3 Department of Thoracic Surgery,First Hospital,Jilin University,Changchun 130021,China)

Received:2006-11-06 Revised:1900-01-01 Online:2007-11-28 Published:2007-11-28
Contact: WANG Zhong-shan

摘要/Abstract

摘要： 目的：包装带有人U6启动子及c-Met短发夹环的重组腺相关病毒，为抑制c-Met的表达及进行肿瘤基因治疗研究奠定基础。方法：通过PCR方法将带有c-Met短发夹结构的片段构建至人U6启动子下游，将U6shMet构建至腺相关病毒载体质粒pSNAV中，将pSNAVUshMet转染BHK细胞，经G418筛选后加辅毒rHSV1-repcap包装成携带U6shMet的重组腺相关病毒。SDS-PAGE电泳分析病毒纯度，斑点杂交测定病毒滴度。结果：获得了2段带有c-Met短发夹结构的片段U6shMet1和U6shMet2,成功包装了2个带有U6shMet序列的重组腺相关病毒rAAVUshMet1和rAAVUshMet2，纯化后的病毒电泳分析条带清晰、杂带少，病毒滴度均为4×10¹²mg•L^-1。结论：成功构建了带有U6shMet的重组腺相关病毒rAAVUshMet，病毒纯度好、滴度高，为抑制c-Met的表达及进行肿瘤基因治疗提供了运载工具。

关键词: 腺相关病毒, RNA干扰

Abstract: To package the recombination adeno-associated virus with human U6 promoter and c-met short hairpin in order to establish foundation for research on inhibiting the expression of c-Met and cancer gene therapy. Methodsc-Met short hairpin was synthesized and linked to the down stream of U6 promoter by PCR. Adeno-associated viral vectors pSNAVUshMet(1,2) were constructed and transfected into BHK cells.Positive cell clones were selected by G418.Adeno-associated virus with U6shMet were packaged by adding rHSV1-repcap.The virus purity was analysed by SDS-PAGE and titer was assayed by dot blot.ResultsTwo fragments (U6shMet1 and U6shMet2) with c-Met short hairpin were obtained and two recombination adeno-associated virus (rAAVUshMet1 and rAAVUshMet2) with U6shMet were packaged successfully.SDS-PAGE analysis showed that the purified virus appeared clear characterized bands.The virus titer was 4×10¹²mg•L^-1. Conclusion The recombination adeno-associated virus (rAAVUshMet1 and rAAVUshMet2) with U6shMetare constructed successfully.The virus have high titer and good purity and could act as the effective vehicle of inhibiting the expression of c-Met and cancer gene therapy.

Key words: adeno-associated virus, RNA interference

中图分类号:

Q786

赵慧，路英丽，王凯忠，王忠山. 携带c-Met短发夹结构重组腺相关病毒的包装及滴度测定[J]. J4, 2007, 33(6): 947-951.

ZHAO Hui,LU Ying-li,WANG Kai-zhong，WANG Zhong-shan. Package and titer assay of recombination adeno-associated viruswith c-Met short hairpin[J]. J4, 2007, 33(6): 947-951.

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携带c-Met短发夹结构重组腺相关病毒的包装及滴度测定

Package and titer assay of recombination adeno-associated viruswith c-Met short hairpin

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