新型双基因表达盒真核载体的构建及功能验证

中国生物工程杂志

2011, Vol. 31

Issue (9): 14-20

研究报告

新型双基因表达盒真核载体的构建及功能验证

杜寿文^1,2, 李昌², 王宇航^1,2, 任大勇^1,2, 刘存霞^1,2, 孙丹丹², 朱娜², 李沂², 秦艳青², 金宁一²

1. 吉林大学畜牧兽医学院长春 130062;
2. 军事医学科学院军事兽医研究所全军基因工程重点实验室长春 130122

Construction and Verification of Neotype Expression Vector Containing Two Expression Cassettes

DU Shou-wen^1,2, LI Chang², WANG Yu-hang^1,2, REN Da-yong^1,2, LIU Cun-xia^1,2, SUN Dan-dan², ZHU Na², LI Yi², QIN Yan-qing², JIN Ning-yi²

1. College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China;
2. Genetic Engineering Laboratory, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China

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摘要：

目的: 构建含双基因表达盒的新型基因疫苗真核表达载体pVAX2,并对其功能进行验证。方法: 设计一个包含人巨细胞病毒启动子(CMV promoter)、T7启动子、信号肽基因、血凝素A表位基因(HA)、多克隆位点区域(MCS)、c-myc抗原表位、血小板来源的生长因子受体跨膜区域(PDGFR TM)及牛生长激素多腺苷酸化信号(BGH polyA)等八种元素的表达盒Ⅱ(Expressing cassette Ⅱ),在其上下游添加Nru Ⅰ酶切位点后,进行人工化学合成,连接到克隆载体pGH中得pGH-Ⅱ;用Nru Ⅰ酶切pGH-Ⅱ,回收1 300bp左右的片段,去磷后插入到pVAX1的相应位点,Nru Ⅰ及Bgl Ⅱ/Pst Ⅰ酶切鉴定,获得新型基因疫苗真核表达载体pVAX2;然后以增强型绿色荧光蛋白(EGFP)为报告基因,将其分别构建至两个不同的表达盒内,脂质体转染BHK-21细胞,利用RT-PCR及荧光显微镜技术进行该载体的功能验证。结果: 两个表达盒内的EGFP基因在BHK-21细胞均能高效表达,相互间不受影响,且新构建的表达盒具有蛋白展示功能。结论: 成功构建含双基因表达盒的新型基因疫苗真核表达载体pVAX2,为多价DNA疫苗的研究奠定了坚实基础。

关键词： pVAX2; EGFP; 双基因表达盒; 功能验证

Abstract:

Objective: To construct and verify the neotype gene vaccine eukaryotic expression vector pVAX2 which contains two gene expression cassettes. Methods: The new expression cassette was designed and obtained by artificial chemical synthesis that contains eight elements—Human cytomegalovirus (CMV) promoter, T7 promoter, signal peptide sequences, Hemagglutinin A epitope genes (HA), multiple cloning sites region(MCS),c-myc epitope, Platelet-derived growth factor receptor transmembrane domain (PDGFR-TM) and Bovine growth hormone polyadenylation (BGH polyA). And the restriction enzyme NruⅠwas added to its upstream and downstream, and then it linked with cloning vector pGH to construct pGH-Ⅱ. The pGH-Ⅱ was cutted with NruⅠ,and the fragment (1 300bp) was reclaimed and cloned into the eukaryotic expression vector pVAX1 after removing phosphoryl groups with Alkaline phosphatase to construct the neotype gene vaccine eukaryotic expression vector pVAX2, which was identified with NruⅠand BglⅡ/PstⅠ. To identify the expression ability of pVAX2, the enhanced green fluorescent protein (EGFP) genes as report gene was inserted into the difference cassette of the two expression cassettes, and then transfected into BHK-21cells by liposome. The function of pVAX2 was verified by the reverse-transcriptase-polymerase chain reaction (RT-PCR) and fluorescence microscope. Result: The expression of EGFP in both expression cassettes was high efficiency in BHK-21 cells, and didn’t impact each other. Especially, the new expression cassette allowed display of proteins on the cell surface. Conclusion: The eukaryotic expression vector pVAX2 is successfully constructed, which could be used for the research of polyvalency DNA vaccines.

Key words: pVAX2 EGFP Two gene expression cassettes Function verification

收稿日期: 2011-05-05 出版日期: 2011-09-25

ZTFLH:

Q786

基金资助:

广西自然科学基金(桂科合0992032-12、桂科自0832152)、广西研究生科研创新项目(2009105981002M180)资助项目

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引用本文:

杜寿文, 李昌, 王宇航, 任大勇, 刘存霞, 孙丹丹, 朱娜, 李沂, 秦艳青, 金宁一. 新型双基因表达盒真核载体的构建及功能验证[J]. 中国生物工程杂志, 2011, 31(9): 14-20.

DU Shou-wen, LI Chang, WANG Yu-hang, REN Da-yong, LIU Cun-xia, SUN Dan-dan, ZHU Na, LI Yi, QIN Yan-qing, JIN Ning-yi. Construction and Verification of Neotype Expression Vector Containing Two Expression Cassettes. China Biotechnology, 2011, 31(9): 14-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I9/14

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