Construction ,Expression and Characteristic Analysis of Recombinant Human TIM-4-EGFP Fusion Protein
10.3870/j.issn.1672-0741.2018.03.002
- VernacularTitle:人类TIM-4-EGFP融合蛋白重组体的构建、表达和特性分析
- Author:
Zhizhong CHEN
1
;
Lihua HU
;
Jilin QIN
Author Information
1. 广西壮族自治区人民医院检验科
- Keywords:
T-cell immunoglobulin mucin-4;
Escherichia coli;
green fluorescent protein;
apoptosis
- From:
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
2018;47(3):262-268
- CountryChina
- Language:Chinese
-
Abstract:
Objective The present study aimed to explore the expression and purification of a fusion protein of human TIM-4 and EGFP in Escherichiacoli(E.coli)and evaluate its bioactivity.Methods The cDNA fragments of human TIM-4 and EG-FP genes were respectively amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a. The constructed re-combinant plasmid pET-28a-TIM-4-EGFP was transformed to E. coli BL21 (DE3)for the expression under the induction of IPTG.The expressed protein was purified by Ni-NTA resin.Recombinant protein was analyzed by SDS-PAGE and Western blotting ,and its binding activity to the apoptotic cells was detected under the fluorescence microscope.Results The TIM-4-EG-FP vector was constructed and expressed in E. coli. The TIM-4-EGFP fusion protein was identified and verified by SDS-PAGE and Western blotting.Our results demonstrated that all the TIM-4-EGFP fusion proteins recognize and bind directly to apoptot-ic cells ,but not to viable cells.We further verified that the interactions of TIM-4-EGFP with apoptotic cells were blocked by TIM-4-Ig fusion proteins.Conclusion We successfully constructed a fusion protein encoding human TIM-4 and EGFP ,and ex-pressed it in E.coli. The fusion protein shows a readily obtainable source of biologically active TIM-4 ,which has considerable potential for further studies on human TIM-4 and its receptor.
