Prokaryotic expression,purification and functional analysis of human RPL22 gene
10.19405/j.cnki.issn1000-1492.2024.10.013
- VernacularTitle:人源性RPL22基因的原核表达与纯化及功能分析
- Author:
Tian TIAN
1
;
Xingjuan ZHANG
;
Mingxia YANG
Author Information
1. 南京医科大学第三附属医院(常州市第二人民医院)呼吸科,常州 213003
- Keywords:
ribosomal protein L22;
non-small cell lung cancer;
bioinformatic analysis;
prokaryotic expression and purification;
functional analysis
- From:
Acta Universitatis Medicinalis Anhui
2024;59(10):1785-1793
- CountryChina
- Language:Chinese
-
Abstract:
Objective To understand the bioinformatics of human ribosomal protein L22(RPL22)gene,prokary-otic expression and purification of human RPL22 recombinant protein,synthesis of human RPL22(labeled as agent M)in vitro,study the effects of agent M on proliferation,cycle and apoptosis of NSCLC A549 cells,and to analyze the effect of combination chemotherapy with M and cisplatin(DDP).Methods RPL22 bioinformatics was ana-lyzed.RPL22 gene was cloned by PCR,the single chain oligonucleotide was designed and synthesized to obtain the target gene,and connect the pET-28α vector.The pET-28α-RPL22 recombinant plasmid was constructed,and the receptive cell DH5α of Escherichia coli was transformed.The expression of recombinant protein RPL22 was analyzed by SDS-PAGE and Western blot.The semi-inhibitory rate(IC50)of M and DDP in A549 cells and the effect of M on the proliferation of A549 cells were detected by CCK-8.The effect of agent M on apoptosis and cycle of A549 cells was detected by flow cytometry.qPCR and Western blot assays were used to detect M and DDP on phosphati-dylinositol-3-kinase/protein kinase B(PI3K/AKT)and adenosine 5'-monophosphate activation of protein kinase/mammalian target of rapamycin(AMPK/mTOR)signaling pathway,respectively.Results RPL22 protein was an unstable and hydrophilic protein.The recombinant protein was dissolved and purified to obtain high concentration of recombinant protein.In A549 cells,the IC50 of reagent M was 400 μg/L,and the IC50 of DDP was 10 μmol/L,and the concentration of reagent M was inversely proportional to the cell activity.The inhibitory effect of reagent M(200 μg/L)combined with DDP(2.5 μmol/L)was similar to that of DDP(10 μmol/L)alone.Reagent M could induce G2 cell cycle arrest and promote apoptosis,and might be involved in the regulation of PI3 K/AKT and AMPK/mTOR pathways.Conclusion The recombinant RPL22 protein is cloned and expressed successfully.
- Full text:2024122512003103821人源性RPL22基因的原核表达与纯化及功能分析_田甜.pdf
