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不同长度poly(A)尾对mRNA体外表达的影响及转录模板在大肠埃希菌中的传代稳定性
马宁
邓涛
张国梅
郑嘉昊
贾兰馨
张家友
杨晓明
作者及单位信息
·
DOI： 10.3760/cma.j.cn112309-20220614-00199
Effects of poly(A) tails with different lengths on mRNA expression in vitro and stability of transcription template in Escherichia coli
Ma Ning
Deng Tao
Zhang Guomei
Zheng Jiahao
Jia Lanxin
Zhang Jiayou
Yang Xiaoming
Authors Info & Affiliations
Ma Ning
The Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products, Wuhan 430207, China
Deng Tao
The Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products, Wuhan 430207, China
Zhang Guomei
The Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products, Wuhan 430207, China
Zheng Jiahao
The Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products, Wuhan 430207, China
Jia Lanxin
The Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products, Wuhan 430207, China
Zhang Jiayou
National Institute of Engineering Technology Research in Combination Vaccine, Wuhan 430207, China
Yang Xiaoming
China National Biotec Group Company Limited, Beijing 100029, China
·
DOI： 10.3760/cma.j.cn112309-20220614-00199
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摘要

目的探索不同长度poly(A)尾对mRNA体外表达的影响以及含poly(A)尾转录模板在大肠埃希菌中的传代稳定性。

方法设计并构建含38、60、103、125 nt和60 nt+6 nt间隔序列+60 nt(简称126 nt)poly(A)尾的模板质粒，利用单酶切将其线性化并以此作为转录模板进行体外转录反应制备增强绿色荧光蛋白(enhanced green fluorescent protein，EGFP)-mRNA。将含有不同长度poly(A)尾的EGFP-mRNA转染293T细胞，流式细胞术检测EGFP表达情况。将poly(A)尾长度为125 nt和126 nt的模板质粒分别转化至大肠埃希菌TransStbl3感受态细胞以及Top10感受态细胞中，各挑取7个单克隆进行培养并提取质粒，质粒经双酶切后进行毛细管电泳检测。分别从中选取3个测序正确的单克隆于37℃条件下进行连续传代，每两代提取一次质粒，双酶切后进行毛细管电泳检测。同时将poly(A)尾长度为125 nt的两组单克隆再分别于30℃条件下进行连续传代，每两代提取一次质粒并进行双酶切鉴定和毛细管电泳检测。

结果成功构建了含不同长度poly(A)尾的转录模板。流式细胞术结果显示，poly(A)尾长度为103 nt和125 nt的模板质粒荧光表达明显高于poly(A)尾长度为38 nt与60 nt的模板质粒。poly(A)尾长度为126 nt的模板质粒荧光表达显著高于其他组。poly(A)尾长度为125 nt的模板质粒转化TransStbl3感受态细胞和Top10感受态细胞后稳定序列的百分比分别为76%和91%，37℃条件下连续传代均可稳定传代至第4代，30℃条件下连续传代可分别稳定传代至第16代和第10代。poly(A)尾长度为126 nt的模板质粒转化TransStbl3感受态细胞和Top10感受态细胞后稳定序列的百分比分别为95%和48%，37℃条件下连续传代均可稳定传代至第12代。

结论mRNA中poly(A)尾的长度和组成对目的蛋白的表达均有影响，在poly(A)尾中添加长度为6 nt的间隔序列以及30℃低温培养均有助于增加模板质粒的传代稳定性，为mRNA疫苗体外转录模板的设计和生产制备提供参考。

Poly(A)尾;mRNA疫苗;转录;表达;稳定性
ABSTRACT

ObjectiveTo investigate the effects of poly(A) tails with different lengths on mRNA expression in vitro and the passage stability of transcription template with poly (A) tail in Escherichia coli (E.coli).

MethodsPlasmids with poly(A) tails of 38, 60, 103, 125 and 126 (60 nt+ 6 nt spacer+ 60 nt) nt were designed and constructed. Then the plasmids were linearized by single enzyme digestion and used as transcription template for preparing enhanced green fluorescent protein (EGFP)-mRNA. EGFP-mRNA containing poly(A) tails of different lengths were transfected into 293T cells and the expression of EGFP was detected by flow cytometry. As to stability test, the template plasmids with poly (A) tail of 125 and 126 nt were transformed into E. coli TransStbl3 and Top10 competent cells. Seven clones were selected for culture and plasmid extraction, and then the plasmids were digested by restriction enzyme and detected by capillary electrophoresis. For passage stability, three correctly sequenced clones of each group were selected for continuous passage at 37℃, and the plasmids were extracted and digested every two generations for capillary electrophoresis. At the same time, the correctly sequenced clones of 125 nt group were also passaged at 30℃, and the plasmids were also extracted and digested every two generations for capillary electrophoresis.

ResultsThe transcription templates with poly(A) tail of different lengths were successfully constructed. Flow cytometry showed that the fluorescence expression of the template plasmids with poly (A) tail of 103 and 125 nt were significantly higher than that of 38 and 60 nt. The fluorescence expression of the plasmid with poly (A) tail of 126 nt was significantly higher than that of all other groups. The percentages of stable sequences of the template plasmid with poly(A) tail of 125 nt in TransStbl3 and Top10 competent cells were 76% and 91%, respectively. The results of continuous passage showed that poly(A) tail of 125 nt could be stable to the 4th generation at 37℃ in both TransStbl3 and Top10 competent cells, and stable to the 16th and 10th generations at 30℃. The percentages of stable sequences of the template plasmid with poly(A) tail of 126 nt in TransStbl3 and Top10 competent cells were 95% and 48%, respectively. The results of continuous passage showed that poly(A) tail of 126 nt could be stable to the 12th generation at 37℃ in both TransStbl3 and Top10 competent cells.

ConclusionsThe length and composition of poly(A) tail in mRNA affected the expression of target protein. Adding a spacer with a length of 6 nt to poly(A) tail and low temperature culture were both helpful to improve the stability of the template plasmid, which provided a reference for the design and preparation of in vitro transcription template of mRNA vaccine.

Poly(A) tail;mRNA vaccine;Transcription;Expression;Stability
Yang Xiaoming, Email: mocdef.mabrahponisgnimoaixgnay, Tel: 0086-27-88843621
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引用本文

马宁,邓涛,张国梅,等. 不同长度poly(A)尾对mRNA体外表达的影响及转录模板在大肠埃希菌中的传代稳定性[J]. 中华微生物学和免疫学杂志,2023,43(01)：47-54.

DOI:10.3760/cma.j.cn112309-20220614-00199

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mRNA疫苗是近年来快速发展起来的一种新型疫苗。该疫苗概念于1990年首次被提出，研究人员发现体外转录制备的mRNA直接注射小鼠骨骼肌细胞后可成功实现目的蛋白的表达[ 1 ]。自此之后，对于mRNA疫苗的结构、功能和递送技术等相关研究得到快速发展。mRNA疫苗作为一种新型疫苗具有许多传统疫苗或DNA疫苗不具备的优势，如设计灵活快速、生产过程不涉及活病毒和细胞培养、工艺易于放大、可同时诱导高水平的细胞免疫和体液免疫应答、无基因组整合风险等[ 2 , 3 ]。因此，在COVID-19疫情暴发期间，mRNA疫苗以其特有的优势快速进入临床并获得美国食品药品监督管理局(Food and Drug Adminstration，FDA)紧急使用授权。截至目前，已有两款新型冠状病毒mRNA疫苗获得FDA批准上市，分别为辉瑞(Pfizer)/BioNTech公司联合开发的BNT162b2以及Moderna公司的mRNA-1273。
典型非复制型mRNA疫苗核酸骨架组成与真核细胞内成熟的mRNA分子类似，均由5′帽子、5′非编码区(5′UTR)、开放阅读框、3′非编码区(3′UTR)和poly(A)尾组成[ 4 ]。通过对各组成部分的优化可显著改善mRNA的翻译效率和稳定性。poly(A)尾是存在于mRNA分子3′末端的一段由重复A组成的序列，该序列的存在一方面可降低RNA核酸外切酶对mRNA分子的降解从而提高分子稳定性，另一方面也可以与poly(A)结合蛋白直接结合并通过翻译起始因子eIF4E介导与5′帽子端相互作用协同调节mRNA的翻译效率和稳定性[ 5 , 6 ]。poly(A)尾长度对mRNA翻译效率有显著影响，但因细胞类型而异[ 7 , 8 ]。例如，研究发现长度为120 nt的poly(A)尾在树突状细胞中具有很好的表达效果[ 9 ]。
体外转录合成的mRNA分子可以有两种加尾方式，一种是在重组poly(A)聚合酶的催化下直接在mRNA分子的3′末端进行加尾，另一种是直接在转录模板中设计添加目标长度的poly(A)尾序列。前者产生的poly(A)尾长度较长但产物不均一，而后者则可以产生固定长度的poly(A)尾，从而更有利于质量控制，因而被广泛应用于生产实践[ 10 ]。尽管如此，仍有许多潜在的问题需要关注，例如在模板DNA中添加大量的重复序列容易发生重组从而导致模板序列的不稳定，因此有必要对模板进行适当设计以提高序列的稳定性。
本研究的主要目的是探索不同长度和组成的poly(A)尾对mRNA表达的影响以及含不同组成poly(A)尾的模板在不同宿主菌和不同温度条件下的传代稳定性，为mRNA疫苗的转录模板设计及制备提供参考。
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备注信息
A
杨晓明，Email：mocdef.mabrahponisgnimoaixgnay，电话：027-88843621
B

马宁：实验设计和操作、数据采集、结果分析和解释、论文撰写和修改；邓涛、张国梅、郑嘉昊、贾兰馨：实验操作、数据采集；张家友、杨晓明：研究指导、论文修改、数据分析、经费支持、行政支持

C
所有作者声明无利益冲突
D
湖北省重点研发计划 (2020DCC002)
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