Prediction and analysis of T/B combined epitope of EM10 protein in Echinococcus multilocularis and identification of expressed products
10.3760/cma.j.cn231583-20240108-00008
- VernacularTitle:棘球蚴EM10蛋白T/B联合表位预测分析及表达产物鉴定
- Author:
Xizhi MA
1
;
Yanmin LI
;
Nafei CHEN
;
Aimaiti ZULIHUMA
;
Jiazhen WANG
;
Xiaotao ZHOU
Author Information
1. 新疆医科大学基础医学院免疫学教研室，乌鲁木齐 830011
- Keywords:
Echinococcosis;
Immunodiagnosis;
Recombinant plasmid
- From:
Chinese Journal of Endemiology
2024;43(10):796-802
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To predict and analyze the T/B combined epitope of EM10 protein in Echinococcus multilocularis, and identify the expressed products of the biosynthetic EM10 multi epitopes. Methods:The gene-related information of EM10 protein was obtained through NCBI GenBank public database. Bioinformatics technique was used to predict and analyze the T/B binding epitopes of EM10 protein. The prokaryotic expession recombinant plasmid pET30a-EM10 (epitope) was synthesized, and transformed into host bacteria Ecoli. BL21 (DE3). The expression of EM10 recombinant multi-epitope protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting after induced expression by isopropyl thiogalactopyranoside (IPTG). Results:The total length of EM10 gene was 1 759 bp (GenBank registration number: U05573), and its protein amino acid sequence (GenBank registration number: AAA50580.1) was 559 amino acids. By using Phyre software for homology modeling, the tertiary structure of EM10 protein was obtained, and the T/B combined epitope of EM10 protein was successfully predicted, the dominant epitope was located at 46 - 61, 133 - 183, 239 - 255 and 442 - 475 amino acid sites. The (GGGGS)n linker sequence was used to connect the epitopes to form an EM10 recombinant multi-epitope protein with a total of 206 amino acid. The size of the DNA fragment was 618 bp and the relative molecular weight of the protein was 22.66 × 10 3. The prokaryotic expession recombinant plasmid was validated by enzyme digestion, the results showed that the plasmid size was between 5 000 and 6 000 bp, which was consistent with the length of the constructed plasmid (5 854 bp). SDS-PAGE showed that the target protein was expressed in the supernatant induced by IPTG at 37 ℃ and the effect was the best. The relative molecular weight of the protein was 22.66 × 10 3 by Western blotting, which was consistent with the constructed plasmid. Conclusions:The combined epitope of EM10 T/B is successfully designed and predicted using bioinformatics technology. A prokaryotic expression recombinant plasmid is constructed, the expression of EM10 recombinant multi-epitope protein is verified through experiments, providing an experimental basis for the construction of an EM10 dominant epitope diagnostic kit.
