鸡EED基因的原核表达与生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.10.005

Abstract

Abstract: This study was aimed to obtain the prokaryotic expression protein of embryonic ectoderm development (EED) in chicken and explore its biological characteristics by bioinformatics. EED gene was amplified from spleen in chicken by RT-PCR, and cloned it into prokaryotic expression vector pET-32a(+) to construct the recombinant expression vector pET-32a-EED. Then pET-32a-EED was transformed into E. coli Rosetta to induce the EED fusion protein expression by IPTG. The recombinant protein was purified, and it was detected by Western blotting. Moreover, the bioinformatics analysis of EED protein in chicken was detected by online software. The results showed that the sequence of EED gene was 1 341 bp, which was 99.85% similarity with the reference sequence (GenBank accession No. :NM_001031376.1). The fusion protein with molecular weight of about 70 ku was obtained when the induction temperature was 28 ℃ and the expression of 1 mmol/L IPTG inducer was added for 7 h. The results of online software analysis showed that the relative molecular weight of EED protein in chicken was 50 377.92, and the isoelectric point was 6.54. It was a hydrophilic protein without transmembrane domain and signal peptide sequence. The tertiary structure prediction of EED protein in chicken was similar to human (6c23.1), and it also had the WD40 domain. In conclusion, the EED soluble protein in chicken was successfully expressed and purified, and EED protein had important functional elementsis, which provided materials for further study on the function of EED protein in chicken.

Key words: chicken; EED gene; expression; purification; bioinformatics

CLC Number:

YU Chuan, SONG Minjie, SHI Shengli, ZHU Wenwen, ZHANG Hewei, TIAN Wenjing, WANG Conghui, CHENG Xiangchao. Prokaryotic Expression and Bioinformatics Analysis of EED Gene in Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3545-3553.

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Prokaryotic Expression and Bioinformatics Analysis of EED Gene in Chicken

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