猪链球菌2型MRP和EF双基因共表达重组腺病毒载体的构建

生物技术通报 ›› 2013, Vol. 0 ›› Issue (8): 130-134.

猪链球菌2型MRP和EF双基因共表达重组腺病毒载体的构建

汪涛^1，2, 余辉¹, 梅钧¹, 李兴暖², 周小鸥², 赵志军³

（1.九江学院基础医学院病原生物学教研室，九江 332000；2.江西省系统生物学临床应用重点实验室，九江 332000；
3.宁夏医科大学总医院医学实验中心，银川 750004）

收稿日期:2013-01-11 修回日期:2013-08-11 出版日期:2013-08-11 发布日期:2013-09-02
作者简介:汪涛，男，博士，研究方向：病原生物分子生物学；E-mail：comwangtaocom@163.com
基金资助:
江西省教育厅科技项目（GJJ12624），九江学院博士科研启动项目（8870909）

Construction of Recombinant Adenovirus Vector of Co-expressing MRP and EF Gene from Streptococcus suis Type 2

Wang Tao^{1, 2}, Yu Hui¹, Mei Jun¹, Li Xingnuan², Zhou Xiaoou², Zhao Zhijun³

（1. Department of Pathogenic Biology，Medical College，Jiujiang University，Jiujiang 332000；2. Key Laboratory of Jiangxi Province for the Systems Biology Clinical Application，Jiujiang 332000；3. Medical Experimentation Center，General Hospital of Ningxia Medical University，Yinchuan 750004）

Received:2013-01-11 Revised:2013-08-11 Published:2013-08-11 Online:2013-09-02

摘要/Abstract

摘要： 旨在构建猪链球菌2型溶菌酶释放蛋白（MRP）和胞外因子（EF）双基因共表达重组腺病毒载体。根据猪链球菌2型MRP和EF的基因序列设计合成2对引物，以猪链球菌2型基因组为模板，扩增MRP基因（第1 801-2 513位）和EF基因（第1 783-2 563位）序列，并克隆到pMD18-T载体。然后将MRP基因、IRES元件和EF基因依次定向克隆至腺病毒穿梭载体pShuttle-CMV中构建重组穿梭质粒pShuttle-CMV-MRP-IRES-EF，再经PmeⅠ酶切，然后转化至含腺病毒骨架质粒 pAdEasy-1的BJ5183-AD-1 感受态细胞中，经同源重组获得重组腺病毒载体质粒rAdeno-CMV-MRP-IRES-EF。PacⅠ酶切线性化该重组腺病毒载体质粒，再转染AD-293细胞进行病毒包装，最后对细胞包装的病毒液进行PCR鉴定。结果表明，克隆的MRP和EF基因测序正确，酶切重组穿梭质粒和腺病毒载体质粒得到特异酶切产物；线性化的重组腺病毒载体质粒rAdeno-CMV-MRP-IRES-EF转染AD-293细胞6 d后也出现明显的细胞病变（CPE），在培养细胞的上清病毒液中也检测到了MRP和EF基因片段。

关键词: 猪链球菌2型, 溶菌酶释放蛋白, 胞外因子, 双基因共表达, 重组腺病毒载体

Abstract: The purpose of this study was to construct recombinant adenovirus vector of co-expression MRP and EF genes from Streptococcus suis type 2（SS2）. The specific primers were designed by the sequence of MRP and EF genes. The MRP（1 801-2 513）and EF（1 783-2 563）gene fragment were amplified from genomic DNA of SS2 by PCR technique. PCR products were cloned in pMD18-T vector. Then MRP gene, IRES element and EF gene linked into the adenovirus shuttle plasmid（pShuttle-CMV）to construct recombinant shuttle plasmid（pShuttle-CMV-MRP-IRES-EF）. The recombinant shuttle plasmid was lineared with PmeⅠand transformed into BJ5183-AD-1 competent cells to construct homogeneous recombinant adenovirus plasmid（rAdeno-CMV-MRP-IRES-EF）. Then the recombinant adenovirus plasmid was linearized with PacⅠand transfected into AD-293 cells. Finally, virus liquids of cell pack were identified by PCR. The results showed that MRP and EF genes were verified by gene sequencing. Both the recombinant shuttle plasmid and recombinant adenovirus vector plasmid were verified by enzyme digestion. Cytopathic effect（CPE）was observed after transfection linear rAdeno-CMV-MRP-EF 6 days. MRP and EF gene were also detected in the virus liquid by PCR.

Key words: Streptococcus suis type 2, Muramidase-released protein, Extracellular factor, Gene co-expression, Recombinant adeno-virus vector

汪涛, 余辉, 梅钧, 李兴暖, 周小鸥, 赵志军. 猪链球菌2型MRP和EF双基因共表达重组腺病毒载体的构建[J]. 生物技术通报, 2013, 0(8): 130-134.

Wang Tao, Yu Hui, Mei Jun, Li Xingnuan, Zhou Xiaoou, Zhao Zhijun. Construction of Recombinant Adenovirus Vector of Co-expressing MRP and EF Gene from Streptococcus suis Type 2[J]. Biotechnology Bulletin, 2013, 0(8): 130-134.

参考文献

[1] van der Velden EW, Raemakers SG, Vecht U, et al. Streptococcus suis type 2 in swine. An imported problem[J]. Tijdschr Diergeneeskd, 1987, 112（11）：660-664.
[2] Fittipaldi N, Segura M, Grenier D, et al. Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis[J]. Future Microbiol, 2012, 7（2）：259-279.
[3] Harel J, Martinez G, Nassar A, et al. Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2[J]. Infect Immun, 2003, 71（10）：6104-6108.
[4] Tang Y, Zhao H, Wu W, et al. Genetic and virulence characterization of Streptococcus suis type 2 isolates from swine in the provinces of Zhejiang and Henan, China[J]. Folia Microbiol（Praha）, 2011, 56（6）：541-548.
[5] 欧瑜, 陆承平.猪链球菌2型溶菌酶释放蛋白基因片段的克隆与表达[J].农业生物技术学报, 2002, 10（1）：72-75.
[6] 徐淑菲, 何孔旺, 陆承平.猪链球菌2型毒力部分片段的克隆表达[J].中国人畜共患病杂志, 2004, 11（20）：943-946.
[7] 胡晓抒, 朱凤才, 汪华, 等.人－猪链球菌感染性综合征研究[J].中华预防医学杂志, 2000, 34（3）：150-152.
[8] Lun ZR, Wang QP, Chen XG, et al. Streptococcus suis：an emerging zoonotic pathogen[J]. Lancet Infect Dis, 2007, 7（3）：201-209.
[9] Jing HB, Yuan J, Wang J, et al. Proteome analysis of Streptococcus suis serotype 2[J]. Proteomics, 2008, 8（2）：333-349.
[10] 欧瑜, 陆承平.猪链球菌2 型国内分离株毒力相关蛋白的分析[J].微生物报, 2002, 42（1）：105-109.
[11] Wisselink HJ, Vecht U, Stockhofe-Zurwieden N, Smith HE. Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine[J]. Vet Rec, 2001, 148（15）：473-477.
[12] Hartman ZC, Appledorn DM, Amalfitano A. Adenovirus vector induced innate immune responses：impact upon efficacy and toxicity in gene therapy and vaccine applications[J]. Virus Res, 2008, 132（1-2）：1-14.
[13] Bru T, Salinas S, Kremer EJ. An update on canine adenovirus type 2 and its vectors[J]. Viruses, 2010, 2（9）：2134-2153.
[14] 曹慧青.多基因共表达载体的构建策略[J].国外医学分子生物学分册, 2002, 24（1）：1-4.