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[1]吴志敏,李光明,白 洁,等.体外构建合成成纤维细胞生长因子-1 mRNA及其表达的初步研究[J].天津医科大学学报,2015,21(05):375-378.
　WU Zhi-min,LI Guang-ming,BAI Jie,et al.Preliminary study of the construction, the synthesis FGF-1 mRNA in vitro and its expression in vivo[J].Journal of Tianjin Medical University,2015,21(05):375-378.
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体外构建合成成纤维细胞生长因子-1 mRNA及其表达的初步研究(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
21卷
期数:
2015年05期
页码:
375-378
栏目:
基础医学
出版日期:
2015-09-20

文章信息/Info

Title:
Preliminary study of the construction, the synthesis FGF-1 mRNA in vitro and its expression in vivo
文章编号:
1006-8147（2015）05-0375-04
作者:
吴志敏; 李光明; 白 洁; 王金鑫; 贾欣雨; 杜晓玲; 朱 泽
(天津医科大学病原生物学教研室，天津300070)
Author(s):
WU Zhi-min; LI Guang-ming; BAI Jie; WANG Jin-xing; JIA Xin-yu; DU Xiao-lin;  ZHU Ze
(Department of Microbiology，Tianjin Medical University，Tianjin 300070，China)
关键词:
体外转录; mRNA稳定性; 成纤维细胞生长因子-1; 体内表达
Keywords:
? in vitro transcription; mRNA stability; FGF1; in vivo expression

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分类号:
Q7
DOI:
-
文献标志码:
A
摘要:
目的：参照mRNA稳定性理论，设计并体外转录合成具有一定稳定性的FGF1 mRNA，对其表达进行初步验证。方法：在pT7TS载体上加poly A、βglobin 3’、5’ UTR，增加GC含量优化设计FGF1序列，并结合mRNA二级结构分析其稳定性，目的序列琼脂糖凝胶电泳及测序验证后，体外转录合成FGF1 mRNA，待浓度及片段大小验证后，行动物实验，通过ELISA法检测FGF1 mRNA表达。结果：二级结构分析显示，优化后FGF1序列稳定性更高；双酶切后琼脂糖凝胶电泳显示pT7TS-FGF1重组载体构建成功，测序验证正确；聚丙烯酰胺凝胶电泳显示合成mRNA大小正确。ELISA检测显示，实验组(92.48 pg/mL)小鼠血清中FGF1蛋白含量较对照组(13.59 pg/mL和15.54 pg/mL)明显升高，差异有统计学意义（P<0.01）；对照组间FGF1蛋白含量接近，二者无统计学差别（P>0.05）。结论：成功构建并体外转录合成稳定的FGF1 mRNA，mRNA与鱼精蛋白结合后回输小鼠在体内成功表达。
Abstract:
?Objective: To optimize and synthesize FGF-1 mRNA by in vitro transcription and investigate its stability and expression both in vitro and in vivo. Methods: Poly A and βglobin 3’ and 5’ UTR on pT7TS were added. FGF1 sequence were optimized by increasing GC content and analyzing its stability with the secondary structure of mRNA. Constructed pT7TS-FGF1 then verified it correction by agarose gel electrophoresis (AGE) and sequencing. After the in vitro transcription FGF1 mRNA, its concentration was measured by Narnodrop and its size was analysed by polyacrylamide gel electrophoresis (PAGE). The expression of mRNA in vivo was detected by ELISA in mice assay. Results: The optimized FGF1 was more stable after being predicted and analyzed by secondary structure. Double digestion verification of agarose gel electrophoresis and the sequencing showed that pT7TS-FGF1 had been constructed successfully. The band on PAGE verified that the mRNA size was correct. ELISA assay result showed the expression of the FGF1 protein in serum in the test group(92.48 pg/mL) was higher than that of the control group(13.59 pg/mL and 15.54 pg/mL ) (P<0.01). No significant difference was found in the expression of the FGF1 protein in serum between the control groups(P>0.05). Conclusion: FGF1 mRN can be successfully constructed and synthesized by in vitro transcription. mRNA bounding with protamine can be expressed in the injected mice.

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备注/Memo

备注/Memo:
基金项目 国家自然科学基金资助项目(81402215)；天津医科大学基金资助项目（2013KYQ13）；

作者简介 吴志敏（1990-），男，硕士在读，研究方向：转录机制及疾病的生物治疗；

通信作者：朱泽，E-mail: zhuze@tijmu.edu.cn。

更新日期/Last Update: 2015-09-21