鸡OC-116基因编码区真核表达载体的筛选及其Real-time PCR检测体系的优化

Screening Eukaryotic Expression Vector for Chicken OC-116 Gene Encoding Region and Optimizing Its Real-time PCR Detection System

实验旨在比较鸡OC-116基因编码区在不同载体上的表达差异,筛选表达量相对较高的重组质粒,同时优化OC-116基因编码区的Real-timePCR体系;制备OC116-GFP、OC116-pcDNA、OC116(M1)-pcDNA和OC116(M1)-GFP4种质粒,分别转染CHO细胞,瞬时表达后制备cDNA、提取总蛋白;然后优化OC-116基因编码区的Real-time PCR体系,并分别通过Real-time PCR、Western blotting鉴定表达水平相对更高的重组质粒。结果表明:优化PCR产物中GC碱基的分布可以排除Real-time PCR反应中熔解曲线双峰问题;更换其他实验空间可以有效解决Real-time PCR反应中的疑似气溶胶污染;pcDNA3.1/myc-His(-)A表达载体诱导OC-116基因编码区翻译表达的能力比pEGFP-N1强;在两类真核表达质粒中,Kozak序列的引入均能明显增强OC-116基因编码区的转录以及翻译水平。结果提示,Real-time PCR反应中双峰熔解曲线以及气溶胶污染可以通过引物设计、更换实验环境进行解决;携带Kozak序列的OC-116(M1)-pcDNA重组质粒目标基因转录、翻译表达水平相对更高,可用于后续研究。

To compare the expression difference of OC-116 gene encoding region in various vectors,and screen out some specific recombinant plasmid with higher expression; simultaneously to optimize the real-time PCR system to detect OC-116 transcripts.Four recombinant plasmids,OC116-GFP,OC116-pcDNA,OC116 （M1）-pcDNA,and OC116 （M1）-GFP,were respectively transfected into the CHO cells for transient expression,and both cDNA and protein were prepared from each treatment; then the real-time PCR system was optimized to detect OC-116 transcripts,and the recombinant plasmid with higher OC-116 expression was identified at both real-time PCR and Western blotting level.The results showed as follows,the issue of double peak dissolution curve in real-time PCR system can be effectively solved through optimizing the GC bases distribution in PCR amplicons; the suspected aerosol disturbance in Real-time PCR system can be effectively solved by using another fresh laboratory; to the recombinant plasmid screening,both recombinant plasmids,which were comprised of OC-116 encoding gene and pcDNA3.1/myc-His （-）A,could more efficiently induce the OC-116 expression than other two recombinant plasmids comprising of OC-116 encoding gene and pEGFP-N1; furthermore,to both expression vectors,no matter pcDNA3.1/myc-His （-）A or pEGFP-N1,the induction of Kozak sequence could more efficiently promote not only transcriptional expression but also translation level for chicken OC-116.In conclusions,the issue of double peak dissolution curve can be effectively resolved through PCR primer designing,and suspected aerosol disturbance can be effectively resolved by using another fresh laboratory; OC-116（M 1）-pcDNA,the recombinant plasmid with Kozak sequence,was capable of promoting OC-116 expression and can be used for further researches.