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

Abstract

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

CLC Number:

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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Construction of Eukaryotic Expression Vectors and Bioinformatics Analysis of Rhoa and Ptgs2 Genes in RAW264.7 Cells

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