细粒棘球绦虫EGR-07243和EGR-07244基因生物信息学分析及原核表达

doi:10.16431/j.cnki.1671-7236.2023.03.001

Abstract

Abstract: 【Objective】 This study was aimed to analyze the bioinformatics properties of two Kunitz-type serine protease inhibitor genes (EGR-07243 and EGR-07244) of Echinococcus granulosus,and perform the prokaryotic expression,so as to detect the immunogenicity of EGR-07243 and EGR-07244 proteins.【Method】 The full-length sequences of EGR-07243 and EGR-07244 genes were amplified by PCR,the physicochemical properties,structural characteristics and phylogenetic relationship of EGR-07243 and EGR-07244 proteins were analyzed by bioinformatics software.The recombinant plasmids pET32a-EGR-07243 and pET32a-EGR-07244 were constructed and transformed into E.coli BL21 (DE3) competent cells.The protein expression was detected by SDS-PAGE,and the antigenicity of the two proteins was analyzed by Western blotting.【Result】 The full-length of EGR-07243 gene was 228 bp,encoding 75 amino acids, the protein molecular formula was C₆₈₆H₁₁₄₆N₂₂₈O₂₉₂S₆₃,the relative molecular mass was about 19 258,containing 3 B cell antigen epitopes,the theoretical isoelectric point was 5.30,the fat coefficient was 17.98,and the hydrophilicity was 0.714,which was a stable protein.The full-length of EGR-07244 gene was 228 bp,encoding 75 amino acids,the protein molecular formula was C₆₇₅H₁₁₂₄N₂₂₈O₂₈₇S₅₆,the relative molecular mass was about 18 800,containing 3 B cell antigen epitopes,the theoretical isoelectric point was 5.32,the fat coefficient was 20.61,and the hydrophilicity was 0.689,which was a stable protein.The amino acid sequence similarity of the two proteins was 72.00%.The amino acid sequence identity of the two domains with Taenia genus were more than 95%,which were closely related.However,and were 46.3% with human,which were far related.Double enzyme digestion and sequencing analysis showed that the recombinant plasmids pET32a-EGR-07243 and pET32a-EGR-07244 were successfully constructed.The recombinant plasmids were transformed into E.coli BL21 (DE3) competent cells,and the expression proteins were successfully induced.Western blotting results showed that the two proteins could be recognized by positive sera of dogs infected with protozoa but not by sera from healthy dogs,indicating that the two proteins had good reactivity.【Conclusion】 EGR-07243 and EGR-07244 proteins could be used as vaccine immune targets in the prevention of fine-grained Echinococcus granulosus infection.The results provided experimental materials for screening candidate molecules of anti-echinococcosis vaccine.

Key words: Echinococcus granulosus; EGR-07243 gene; EGR-07244 gene; bioinformatics analysis; prokaryotic expression

CLC Number:

S852.73⁺4

JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.

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Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus

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