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

摘要/Abstract

摘要： 【目的】分析细粒棘球绦虫(Echinococcus granulosus)2个Kunitz型丝氨酸蛋白酶抑制剂基因EGR-07243和EGR-07244的生物信息学特性,并进行原核表达,以期检测EGR-07243和EGR-07244蛋白的免疫原性。【方法】利用PCR扩增EGR-07243、EGR-07244基因全长序列,采用生物信息学软件对EGR-07243、EGR-07244蛋白的理化性质、结构特点及亲缘进化关系进行分析;构建重组质粒pET32a-EGR-07243、pET32a-EGR-07244并转化大肠杆菌BL21(DE3)感受态细胞,利用SDS-PAGE检测蛋白的表达,并通过Western blotting对蛋白进行抗原性分析。【结果】EGR-07243基因全长228 bp,编码75个氨基酸,蛋白分子式为C₆₈₆H₁₁₄₆N₂₂₈O₂₉₂S₆₃,相对分子质量约19258,含有3个B细胞抗原表位,理论等电点为5.30,脂肪系数为17.98,亲水性为0.714,为稳定蛋白;EGR-07244基因全长228 bp,编码75个氨基酸,蛋白分子式为C₆₇₅H₁₁₂₄N₂₂₈O₂₈₇S₅₆,相对分子质量约18800,含有3个B细胞抗原表位,理论等电点为5.32,脂肪系数为20.61,亲水性为0.689,为稳定蛋白。二者氨基酸序列相似性为72.00%。二者与绦虫属的功能结构域氨基酸序列一致性为95%以上,亲缘关系极近;而与人的功能结构域氨基酸序列一致性为46.3%,亲缘关系较远。通过双酶切鉴定及测序分析证明,pET32a-EGR-07243、pET32a-EGR-07244重组质粒构建成功。重组质粒转化大肠杆菌BL21(DE3)感受态细胞后成功诱导表达蛋白,Western blotting结果显示,2种蛋白能被感染原头蚴的犬阳性血清识别且不被健康犬血清识别,表明其均具有良好的反应原性。【结论】EGR-07243和EGR-07244蛋白能够作为预防细粒棘球绦虫感染的疫苗免疫靶点,结果为抗包虫病疫苗候选分子的筛选提供了试验材料。

关键词: 细粒棘球绦虫; EGR-07243基因; EGR-07244基因; 生物信息学分析; 原核表达

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

中图分类号:

S852.73⁺4

贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 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[J]. 中国畜牧兽医, 2023, 50(3): 847-858.

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.

参考文献

[1] 张文宝,张壮志,郑雪婷,等.棘球蚴(包虫)病预防疫苗的研制与应用[J].中国人兽共患病学报,2018, 34(9):834-838. ZHANG W B,ZHANG Z Z,ZHENG X T,et al.Development and application of vaccines against echinococcosis[J].Chinese Journal of Zoonoses,2018,34(9):834-838.(in Chinese)
[2] DEPLAZES P,RINALDI L,ROJAS C,et al.Global distribution of alveolar and cystic echinococcosis [J]. Advances in Parasitology,2017,95:315-493.
[3] 王正荣,朱兴全,贾万忠,等.包虫病的流行危害和防控技术[J].现代畜牧兽医,2019,6:44-48. WANG Z R,ZHU X Q,JIA W Z,et al.Epidemic hazards and prevention and control techniques of echinococcosis[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2019,6:44-48.(in Chinese)
[4] TURRÀ D,VITALE S,MARRA R,et al.Heterologous expression of PKPI and Pin1 proteinase inhibitors enhances plant fitness and broad-spectrum resistance to biotic threats[J].Frontiers in Plant Science,2020,11:461.
[5] DO AMARAL M,FREITAS A C O,SANTOS A S,et al.TcTI,a Kunitz-type trypsin inhibitor from cocoa associated with defense against pathogens[J].Scientific Reports,2022,12(1):698.
[6] CID-GALLEGOS M S,CORZO-RÍOS L J,JIMÉNEZ-MARTÍNEZ C,et al.Protease inhibitors from plants as therapeutic agents—A review [J].Plant Foods for Human Nutrition,2022,77(1):20-29.
[7] 朱艳,高如秀,雷蕾,等.美洲钩虫Ascris型蛋白酶抑制剂1的特征鉴定[J].热带医学杂志,2021,21(5):531-534. ZHU Y,GAO R X,LEI L,et al.Characterization of Ascris-type protease inhibitor 1 from Necator americanus[J].Journal of Tropical Medicine,2021,21(5):531-534.(in Chinese)
[8] 吴杰.捻转血矛线虫Kunitz型丝氨酸蛋白酶抑制因子Hc-spi-i2基因的特性研究[D].杭州:浙江大学,2021. WU J.Characterization of Kunitz-type serine protease inhibitor Hc-spi-i2 gene of Haemonchus contortus[D].Hangzhou:Zhejiang University,2021.(in Chinese)
[9] 马静,高金亮.蜱Kunitz型蛋白酶抑制剂的抗凝机制研究进展[J].中国媒介生物学及控制杂志,2021,32(1):111-114. MA J,GAO J L.Research advances in anticoagulant mechanisms of tick-derived protease inhibitors containing the Kunitz domain[J].Chinese Journal of Vector Biology and Control,2021,32(1):111-114.(in Chinese)
[10] RANASINGHE S L,FISCHER K,ZHANG W B,et al.Cloning and characterization of two potent Kunitz type protease inhibitors from Echinococcus granulosus [J].PLoS Neglected Tropical Diseases,2015,9:e0004268.
[11] ZHANG H,TIAN M,QI W,et al. Bioinformatic comparison of Kunitz protease inhibitors in Echinococcus granulosus sensu stricto and E.multilocularis and the genes expressed in different developmental stages of E.granulosus s.s [J]. BMC Genomics,2021,22:907.
[12] KUMAR S,STECHER G,TAMUR A.MEGA 7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,2016,33:1870-1874.
[13] CHEN C J,CHEN H,ZHANG Y,et al.TBtools:An integrative toolkit developed for interactive analyses of big biological data[J].Molecular Plant,2020,13:1194-1202.
[14] CHUDZINSKI-TAVASSI A M,DE-SÁ-JÚNIOR P L,SIMONS S M,et al.A new tick Kunitz type inhibitor,Amblyomin-X,induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system [J].Toxicon,2010,56:1145-1154.
[15] 刘云扬,蒋帅,李谦,等.Kunitz型丝氨酸蛋白酶抑制剂研究进展[J].生物工程学报,2021,37(11):3988-4000. LIU Y Y,JIANG S,LI Q,et al.Advances of Kunitz-type serine protease inhibitors[J].Chinese Journal of Biotechnology,2021,37(11):3988-4000.(in Chinese)
[16] KAWAI T,AKIRA S.The role of pattern-recognition receptors in innate immunity:Update on Toll-like receptors[J].Nature Immunology,2010,11:373-384.
[17] DE MAGALHÃES M T Q,MAMBELLI F S,SANTOS B P O,et al.Serine protease inhibitors containing a Kunitz domain:Their role in modulation of host inflammatory responses and parasite survival[J].Microbes and Infection,2018,20(9/10):606-609.
[18] WALSH K P,BRADY M T,FINLAY C M,et al.Infection with a helminth parasite attenuates autoimmunity through TGF-beta-mediated suppression of Th17 and Th1 responses[J]. The Journal of Immunology,2009,183:1577-1586.
[19] FALCÓN C R,MASIH D,GATTI G,et al.Fasciola hepatica Kunitz type molecule decreases dendritic cell activation and their ability to induce inflammatory responses[J].PLoS One,2014,9(12):e114505.
[20] ZHANG W Z,LI L,ZHENG Y,et al.Schistosoma japonicum peptide SJMHE1 suppresses airway inflammation of allergic asthma in mice[J].Journal of Cellular and Molecular Medicine,2019,23(11):7819-7829.
[21] GRETEN F R,GRIVENNIKOV S I.Inflammation and cancer:Triggers,mechanisms,and consequences[J].Immunity,2019,51:27-41.
[22] SHIGETOMI H,ONOGI A,KAJIWARA H,et al.Anti-inflammatory actions of serine protease inhibitors containing the Kunitz domain[J].Inflammation Research,2010,59:679-687.
[23] BAYRAKTAR E,IGCI M,ERTURHAN S,et al.Reduced gene expression of bikunin as a prognostic marker for renal cell carcinoma[J].Experimental Oncology,2014,36:107-111.
[24] MORAIS K L,PACHECO M T,BERRA C M,et al.Amblyomin-X induces ER stress,mitochondrial dysfunction,and caspase activation in human melanoma and pancreatic tumor cell[J].Molecular and Cellular Biochemistry, 2016,415:119-131.
[25] PACHECO M T,MORAIS K L,BERRA C M,et al.Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule,Amblyomin-X[J].Experimental Cell Research,2016,340:248-258.