摘要
目的:参照m RNA稳定性理论,设计并体外转录合成具有一定稳定性的FGF1 m RNA,对其表达进行初步验证。方法:在p T7TS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m RNA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 m RNA,待浓度及片段大小验证后,行动物实验,通过ELISA法检测FGF1 m RNA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p T7TS-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m RNA大小正确。ELISA检测显示,实验组(92.48 pg/m L)小鼠血清中FGF1蛋白含量较对照组(13.59 pg/m L和15.54 pg/m L)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 m RNA,m RNA与鱼精蛋白结合后回输小鼠在体内成功表达。
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 β globin 3＇ and 5＇ UTR on pT-/TS were added. FGF1 sequence were optimized by increasing GC content and analyzing its stability with the secondary structure of mRNA. Constructed pTTTS-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 eleetrophoresis （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 pTTTS-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 mRNA can be successfully constructed and synthesized by in vitro transcription, mRNA bounding with protamine can be expressed in the injected mice.
出处
《天津医科大学学报》
2015年第5期375-378,384,共5页
Journal of Tianjin Medical University
基金
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)
