表达β-珠蛋白基因的安全性慢病毒载体的优化 <sup>*</sup>

doi:10.13523/j.cb.20180707

中国生物工程杂志

2018, Vol. 38

Issue (7): 50-57 DOI: 10.13523/j.cb.20180707

技术与方法

表达β-珠蛋白基因的安全性慢病毒载体的优化 ^*

韩亚丽¹,杨冠恒^1,²,陈雁雯¹,龚秀丽¹,张敬之^1,^2,^**(

)

1 上海交通大学附属儿童医院上海市儿童医院上海交通大学医学遗传研究所上海 200040
2 卫生部医学胚胎与分子生物学重点实验室上海市胚胎与生殖工程重点实验室上海 200040

The Optimization of Self-deleting Lentiviral Vector Carrying Human β-globin Gene and Promoter

Ya-li HAN¹,Guang-heng YANG^1,²,Yan-wen CHEN¹,Xiu-li GONG¹,Jing-zhi ZHANG^1,^2,^**(

)

1 Shanghai Institute of Medical Genetics, Children’s Hospital of Shanghai, Children’s Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 20040, China;
2 Key Laboratory of Embryo Molecular Biology, Ministry of Health, Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 20040, China

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摘要：

目的:慢病毒载体(lentiviral vector,LVV)是一种有效的基因治疗导入系统。拟用已研发的携带人的β-珠蛋白基因自删除慢病毒载体,优化其表达有效性和提高其病毒颗粒数。方法:比较三款不同的启动子预测软件的分析结果,分别构建三种不同长度启动子的表达β-珠蛋白基因(β-globin)的LVV,并对其Ⅱ号内含子进行部分删减;用经优化的LVV转导β-地中海贫血(β-地贫)的小鼠诱导性多能性干细胞(induced pluripotent stem cells,iPSC)后,用此iPSC制备嵌合体小鼠模型;经RT-PCR、血涂片瑞氏吉姆萨染色等观察分析其功能性代偿的潜能。研究结果:经优化后的自删除慢病毒载体病毒对其病毒颗粒数的滴度影响不大(2.3×10¹¹LPs/ml),可在嵌合体小鼠模型体内检测到正常人β-珠蛋白基因的功能性表达。结论优化了表达人β-珠蛋白基因的自删除LVV。

关键词： β-地中海贫血; 自删除慢病毒载体; 优化; 基因治疗

Abstract:

Objective: Optimization of previous developed self-deleting lentiviral vector carrying human β-globin gene and promoter in terms of preparation titles and transgene expressing efficacy. Methods: Based on the comparison of the predictions of three on line promoter prediction softwares; three self-deleting lentiviral vectors carrying three different lengths of β-globin promoters with gene were constructed. The optimization was carried out in terms of preparation titles and transgene expressing efficacy. The optimized LVV was used to transduce murine -thalassemia iPSC;and resulted cells were used to generate chimeric mice. RT-PCR and Wright Giemsa staining were then carried out on the mice blood. Results: The viral particles prepared by the optimized LVV principally have no difference comparing with their parental virus, FUGW. The functional expression of normal spliced human β-globin gene was detected in the chimeric mice. And the morphologically normal of erythrocytes was observed. Conclusion: An optimized self-deletion lentiviral vector, FCB-P265, was made.

Key words: β-thalassemia Self-deleted lentivirus vector Optimization Gene therapy

收稿日期: 2018-03-22 出版日期: 2018-08-13

ZTFLH:

Q819

基金资助: 国家自然科学基金面上项目(81570172)

通讯作者: 张敬之 E-mail: jzhang38@hotmail.com

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引用本文:

韩亚丽,杨冠恒,陈雁雯,龚秀丽,张敬之. 表达β-珠蛋白基因的安全性慢病毒载体的优化 ^*[J]. 中国生物工程杂志, 2018, 38(7): 50-57.

Ya-li HAN,Guang-heng YANG,Yan-wen CHEN,Xiu-li GONG,Jing-zhi ZHANG. The Optimization of Self-deleting Lentiviral Vector Carrying Human β-globin Gene and Promoter. China Biotechnology, 2018, 38(7): 50-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180707 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/50

图1 自删除慢病毒载体的工作原理

表1 引物列表

表2 不同的启动子预测软件对人β-globin基因启动子的预测结果

图2 启动子生物信息学分析结果结构示意图

图3 三种慢病毒载体结构图

图4 三种不同的拟病毒载体的结构图

图5 不同的拟病毒载体表达β-globin基因效果比较

图6 不同启动子长度的自删除慢病毒载体包装病毒颗粒数比较

图7 FCB-P265慢病毒成功感染并整合入β-地贫iPS细胞

图8 嵌合体模型小鼠体内外源基因的表达及其红细胞质量

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