人Semaphorin 4D慢病毒载体的构建及鉴定

中国生物工程杂志

2011, Vol. 31

Issue (7): 1-7

研究报告

人Semaphorin 4D慢病毒载体的构建及鉴定

孙强明, 潘玥, 赵玉娇, 陈俊英, 施海晶, 马绍辉

北京协和医学院/中国医学科学院医学生物学研究所云南省重大传染病疫苗研发重点实验室昆明 650118

Construction and Identification of a Lentiviral Vector Expressing Semaphorin 4D

SUN Qiang-ming, PAN Yue, ZHAO Yu-jiao, CHEN Jun-ying, SHI Hai-jing, MA Shao-hui

Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming 650118, China

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摘要：

目的:构建并制备能够有效表达Semaphorin 4D的重组慢病毒。方法:从人急性T细胞白血病Jurkat细胞DNA 扩增人Semaphorin 4D基因,克隆至pWPI GW慢病毒载体上,与pVSVG及pSPAX质粒共转染人胚肾293T细胞,包装出重组慢病毒,将纯化后的重组病毒直接感染293T和HUVEC细胞,通过免疫印迹、免疫荧光染色和血管内皮细胞迁移分析等方法检测Semaphorin 4D的表达和诱导血管内皮细胞迁移的作用。结果: 重组慢病毒介导Semaphorin 4D在293T和HUVEC内获得表达,能介导血管内皮细胞迁移。结论:成功构建了表达Semaphorin 4D的重组慢病毒载体。

关键词： Semaphorin 4D; 慢病毒; 血管新生; 基因治疗

Abstract:

Objective: To construct a lentiviral vector expressing Semaphorin 4D to investigate the role of Semaphorin 4D in angiogenesis. Methods: Semaphorin 4D sequence was amplified from the Jurkat cell cDNA, inserted into pWPI GW vector and identified by DNA sequencing. The packing cell line (human embryonic kidney 293T cell) was cotransfected with pWPI GW/Sema4D, pVSVG and pSPAX. The recombinant lentivirus was packaged and amplified, followed by infection of 293T and HUVEC cells. The expression level of Semaphorin 4D gene was analyzed by Western blot and immunofluresence staining. A Boyden chamber migration assay was performed to establish the biological significance of Semaphorin 4D. Results: The recombinant lentiviral vector expressing Semaphorin 4D was successfully constructed. Western blot and immunofluresence staining results demonstrated that Semaphorin 4D was successfully expressed in 293T and HUVEC cells infected by recombinant lentivirus expressing Semaphorin 4D. Boyden chamber migration assay results showed that enhanced endothelial cell migration is induced by Sema4D-expressing Hela cells.

Key words: Semaphorin 4D Lentivirus Angiogenesis Gene therapy

收稿日期: 2011-02-21 出版日期: 2011-07-25

ZTFLH:

Q819

基金资助:

国家自然科学基金(30571355)、广东省自然科学基金(9451018201003601)资助项目

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引用本文:

孙强明, 潘玥, 赵玉娇, 陈俊英, 施海晶, 马绍辉. 人Semaphorin 4D慢病毒载体的构建及鉴定[J]. 中国生物工程杂志, 2011, 31(7): 1-7.

SUN Qiang-ming, PAN Yue, ZHAO Yu-jiao, CHEN Jun-ying, SHI Hai-jing, MA Shao-hui. Construction and Identification of a Lentiviral Vector Expressing Semaphorin 4D. China Biotechnology, 2011, 31(7): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I7/1

[1] Swiercz J M, Kuner R, Behrens J, et al. Plexin-B1 Directly Interacts with PDZ-RhoGEF/LARG to Regulate RhoA and Growth Cone Morphology. Neuron, 2002, 35(1):51-63.

[2] Conrotto P, Valdembri D, Corso S,et al. Sema4D induces angiogenesis through Met recruitment by Plexin B1. Blood, 2005, 105(11):4321-4329.

[3] Basile J R, Barac A, Zhu T, et al. Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B. Cancer Res, 2004, 64(15):5212-5224.

[4] Sierra J R, Corso S, Caione L, et al. Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages. J Exp Med, 2008, 205(7):1673-1685.

[5] Basile J R, Castilho R M, Williams V P, et al. Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis. Proc Natl Acad Sci USA, 2006, 103(24):9017-9022.

[6] Sun Q M, Nawabi-Ghasimi F, Basile J R. Semaphorins in vascular development and head and neck squamous cell carcinoma-induced angiogenesis. Oral Oncol, 2008, 44(6):523-531.

[7] Matrai J, Chuah M K, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther, 2010, 18(3):477-490.

[8] Tiscornia G, Singer O, Verma I M. Production and purification of lentiviral vectors. Nat Protoc, 2006, 1(1):241-245.

[9] Kolodkin A L, Matthes D J, Goodman C S. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell, 1993, 75(7):1389-1399.

[10] Dittgen T, Nimmerjahn A, Komai S, et al.Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci USA, 2004, 101(52):18206-18211.

[11] Cockrell A S, Kafri T.Gene delivery by lentivirus vectors. Mol Biotechnol, 2007, 36(3):184-204.

[12] Cartier N, Hacein-Bey-Abina S, Bartholomae C C, et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science, 2009, 326(5954):818-823.

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