体外靶向筛选小鼠精子特异性PRSS37基因最适siRNA  

In vitro targeted screening of optimal siRNA for mouse sperm-specific PRSS37 gene

作　　者：高斯 赵萍 刘晓雅 方桂杰 任红艳[2] 毕延震[2] GAO Si;ZHAO Ping;LIU Xiaoya;FANG Guijie;REN Hongyan;BI Yanzhen(Key Laboratory of Fermentation Engineering,Ministry of Education,College of Biological Engineering and Food,Hubei University of Technology,Wuhan 430068;Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding,Institute of Animal Science and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan 430064,China)

机构地区：[1]湖北工业大学生物工程与食品学院发酵工程教育部重点实验室,湖北武汉430068 [2]湖北省农业科学院畜牧兽医研究所动物胚胎工程及分子育种湖北省重点实验室,湖北武汉430064

出　　处：《生物技术》2025年第1期20-24,共5页Biotechnology

基　　金：湖北省中央引导地方科技发展资金项目(2022BGE231);湖北省国际科技合作项目(2021EHB023);中国科学院战略性先导科技专项(XDA24030204)。

摘　　要：[目的]细胞水平靶向筛选出小鼠精子特异性PRSS37基因的最适siRNA序列。[方法]构建PRSS37过表达载体pcDNA3.1-Kozak-3×Flag-PRSS37,并将其转染至GC-2细胞,通过Western Blot检测PRSS37的异位表达。基于PRSS37序列设计并合成3对siRNA序列,将PRSS37过表达载体和siRNA共转染于GC-2细胞,实时荧光定量PCR检测siRNA对PRSS37干扰效率,筛选最有效的siRNA。[结果]PRSS37过表达载体在GC-2细胞中成功表达,与对照组相比,实验组中PRSS37蛋白表达显著升高;设计合成的3对siRNA均能有效干扰PRSS37的表达,与未干扰组相比差异显著(P<0.05),其中siRNA-1干扰效率最高,干扰效率约为63%。[结论]成功实现了PRSS37在GC-2细胞中的异位表达,并在细胞水平筛选出小鼠PRSS37最适siRNA序列。[Objective]Targeted selection of the optimal siRNA sequence for mouse sperm-specific PRSS37 gene at the cellular level.[Method]The PRSS37 overexpression vector pcDNA3.1-Kozak-3×Flag-PRSS37 was constructed,and the overexpression vector was transfected into GC-2 cells to detect the ectopic expression of PRSS37 by Western Blot.Three pairs of siRNA sequences were designed and synthesized based on PRSS37,and PRSS37 overexpression vector and siRNA were co-transfected into GC-2 cells,and the interference efficiency of siRNA on PRSS37 was detected by real-time PCR to select the most effective siRNA.[Result]PRSS37 overexpression vector was successfully expressed in GC-2 cells,and the protein expression of PRSS37 was significantly increased in the experimental group compared with the control group;all three pairs of siRNAs designed and synthesized could effectively interfere with the expression of PRSS37,with significant differences compared with the uninterfered group(P<0.05),with siRNA-1 having the best interference efficiency and interference efficiency of about 63%.[Conclusion]The ectopic expression of PRSS37 in GC-2 was successfully achieved,and the optimal siRNA sequence for mouse PRSS37 was selected at the cellular level.

关 键 词：丝氨酸蛋白酶37 表达载体 异位表达 SIRNA 共转染 干扰效率 

分 类 号：Q786[生物学—分子生物学]