RAW264.7细胞Rhoa和Ptgs2基因真核表达载体构建及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.07.005

摘要/Abstract

摘要： 为深入研究Ras同源基因家族成员A（Ras homolog gene family，member A，Rhoa）和环氧合酶2（cyclooxygenase 2，Ptgs2）分子在布鲁菌逃逸机体免疫中发挥的作用，试验对RAW264.7细胞Rhoa和Ptgs2基因进行扩增与克隆，构建真核表达载体并预测生物信息学功能。根据GenBank数据库中公布的RAW264.7细胞Rhoa和Ptgs2基因CDS区序列（登录号：JN971019.1和NM_011198）设计引物，提取RAW264.7细胞总RNA并反转录为cDNA，经RT-PCR扩增Rhoa和Ptgs2片段并测序，将纯化的Rhoa和Ptgs2片段分别与线性化pcDNA3.1质粒相连接，对重组质粒进行测序分析和双酶切鉴定后，利用Lipofectamine^TM 2000转染293T细胞，经实时荧光定量PCR和Western blotting验证Rhoa和Ptgs2基因的表达情况，并应用生物信息学软件对Rhoa和Ptgs2基因进行预测分析。结果显示，试验成功构建了RAW264.7细胞Rhoa和Ptgs2基因的真核表达质粒；实时荧光定量PCR检测均在转录水平上表达；Western blotting可见Ptgs2蛋白在70 ku处有一明显条带，而Rhoa蛋白未出现条带。生物信息学预测显示，Rhoa和Ptgs2基因核苷酸序列相似性较高，在不同物种之间较为保守；Rhoa不具有信号肽，为不稳定蛋白，而Ptgs2在第17-18位氨基酸处存在信号肽，为稳定蛋白；Rhoa和Ptgs2蛋白分别有12和53个潜在的磷酸化位点；二级结构、三级结构均以无规则卷曲为主。本研究成功构建了RAW264.7细胞Rhoa和Ptgs2基因的真核表达载体，均在转录水平上表达，并分析了其生物学功能，为后续开展RAW264.7细胞Rhoa和Ptgs2基因在布鲁菌免疫机制方面的研究提供了工具。

关键词: RAW264.7细胞; Rhoa基因; Ptgs2基因; 真核表达载体; 生物信息学

Abstract: To explore the role of Ras homolog gene family,member A (Rhoa) and cyclooxygenase 2 (Ptgs2) molecules in the immune escape of Brucella,Rhoa and Ptgs2 genes of RAW264.7 cells were amplified and cloned to construct eukaryotic expression vectors and predict bioinformatics functions.The primers were designed according to Rhoa and Ptgs2 genes CDS sequences published in GenBank (accession No.:JN971019.1 and NM_011198).The total RNA of RAW264.7 cells was extracted and reverse transcribed into cDNA.The purified Rhoa and Ptgs2 fragments were respectively connected to the linearized pcDNA3.1 plasmid,and the recombinant plasmid was sequenced and identified by double enzyme digestion,and then transfected with Lipofectamine^TM 2000 to 293T cells,the expression of Rhoa and Ptgs2 genes was verified by Real-time quantitative PCR and Western blotting.Bioinformatics software was used to predict Rhoa and Ptgs2 genes.The results showed that the eukaryotic expression plasmids of Rhoa and Ptgs2 genes in RAW264.7 cells were successfully constructed,and was expressed at the transcription level by Real-time quantitative PCR detection.Western blotting results showed that Ptgs2 protein had an obvious band at 70 ku,while Rhoa protein had no band.Bioinformatics analysis results showed that the nucleotide sequences of Rhoa and Ptgs2 genes had high similarity and were relatively conserved among different species.RhoA had no signal peptide and was an unstable protein,while Ptgs2 had a signal peptide at 17-18 amino acids position and was a stable protein.Rhoa and Ptgs2 proteins had 12 and 53 potential phosphorylation sites,respectively,and the secondary and tertiary structures were mainly random coil.In this study,the eukaryotic expression vectors of Rhoa and Ptgs2 genes in RAW264.7 cells were successfully constructed,both were expressed at the transcription level,and their biological functions were analyzed,which provided a tool for the follow-up study of Rhoa and Ptgs2 genes in RAW264.7 cells in the immune mechanism of Brucella.

Key words: RAW264.7 cells; Rhoa gene; Ptgs2 gene; eukaryotic expression vector; bioinformatics

中图分类号:

乔连江, 陈晨, 张如, 崔立恒, 徐丽, 杨艳玲. RAW264.7细胞Rhoa和Ptgs2基因真核表达载体构建及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(7): 2313-2323.

QIAO Lianjiang, CHEN Chen, ZHANG Ru, CUI Liheng, XU Li, YANG Yanling. Construction of Eukaryotic Expression Vectors and Bioinformatics Analysis of Rhoa and Ptgs2 Genes in RAW264.7 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2313-2323.

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