目的:应用基因工程技术和生物信息学方法对结核分枝杆菌GroEL1进行原核表达与纯化及其结构和功能预测,分析该抗原在新型结核疫苗的应用价值。 方法:采用PCR技术体外扩增GroEL1基因,并克隆至pET28a质粒中,测序筛选构建成功的pET28a-GroEL1载体,将其载体转化至大肠杆菌BL21(DE3)表达菌株中,IPTG诱导表达重组GroEL1蛋白,利用镍亲和层析柱纯化。从UniProt数据库中获取H37Ra株GroEL1核苷酸和氨基酸序列;分别运用Protparam、TMHMM-2.0、Protscale、NetChop-3.1、Psortb、SignalP-4.1工具预测GroEL1蛋白的理化性质、跨膜螺旋、亲/疏水性、磷酸化位点、亚细胞定位,信号肽;NetNGlyc-1.0和YinOYang-1.2预测其糖基化位点;SOPMA和Swissmodel预测蛋白的二级结构和三级结构;Clustalw对同源序列进行比对。 String预测相互作用蛋白,IEBD和ABCpred预测蛋白的B细胞抗原表位。结果:成功构建重组pET28a-GroEL1载体,GroEL1蛋白在大肠杆菌中部分以可溶形式表达。镍亲和层析柱纯化重组GroEL1蛋白,其纯度达90%以上。Western blot鉴定证实重组GroEL1蛋白具有良好的免疫反应性。结核分枝杆菌H37Ra株GroEL1基因全长1 620 bp,编码539个氨基酸,分子量55.88 kD,等电点为4.98,预测显示该蛋白亲水性较强、性质稳定,无跨膜区,有37个可能的磷酸化修饰位点和8个O-糖基化位点,属于非分泌蛋白,定位在细胞质,二级结构显示α-螺旋占53.43%,延伸链占11.87%,β-转角占7.61%,无规则卷曲占27.09%,有多个B细胞抗原表位及多个与GroEL1蛋白相互作用蛋白。结论:成功表达并纯化重组GroEL1蛋白,运用生物信息学成功预测GroEL1蛋白的结构和功能,为结核病的预防、诊断和治疗奠定基础。
Objective:To study the prokaryotic expression and purification of GroEL1 from mycobacterium tuberculosis by genetic engineering and bioinformatics, and to predict its structure and function, and to analyze its application value in novel tuberculosis vaccine. Methods:The GroEL1 gene was amplified by PCR in vitro and cloned into the pET28a plasmid. The pET28a-GroEL1 vector was successfully constructed by sequencing and transformed into the expression strain of E. coli BL21 (DE3). The recombinant GroEL1 protein was expressed by IPTG and purified by nickel affinity column. The nucleotide and amino acid sequences of GroEL1 of H37Ra strain were obtained from UniProt database. Protparam, TMHMM-2.0, Protscale, NetChop-3.1, Psortb and SignalP-4.1 were used to predict the physicochemical properties, transmembrane helix, hydrophilic/hydrophobic, phosphorylation sites, subcellular localization and signal peptide of GroEL1 protein, respectively. NetNGlyc-1.0 and YinOYang-1.2 predicted the glycosylation sites. SOPMA and Swissmodel predicted the secondary structure and tertiary structure of the protein. Clustalw compares homologous sequences. String predicted interacting proteins, IEBD and ABCpred predicted B-cell epitopes of proteins. Results:The recombinant pET28a-GroEL1 vector was successfully constructed and the GroEL1 protein partially expressed in soluble form in E. coli. The recombinant GroEL1 protein was purified by nickel affinity chromatography with a purity of more than 90%. Western blot analysis confirmed that the recombinant GroEL1 protein had good immunoreactivity. The GroEL1 gene of mycobacterium tuberculosis strain H37Ra has a total length of 1 620 bp, encoding 539 amino acids with a molecular weight of 55.88 kD and an isoelectric point of 4.98. It is predicted that the protein has strong hydrophilicity, stable properties, no transmembrane region, 37 possible phosphorylation modification sites and 8 O-glycosylation sites, which is a non-secreted protein. Located in the cytoplasm, the secondary structure showed α-helix (53.43%), extended chain (11.87%), β-turn(7.61%), random coil(27.09%), multiple B cell antigen epitopes and multiple GroEL1 protein interacting proteins. Conclusion:The recombinant GroEL1 protein was successfully expressed and purified, and the structure and function of GroEL1 protein were predicted by bioinformatics, which laid a foundation for the prevention, diagnosis and treatment of tuberculosis.
[1] Bagcchi S. WHO's global tuberculosis report 2022[J]. Lancet Microbe, 2023, 4(1): e20.
[2] 何磊, 张鹭, 彭超, 等. 结核分枝杆菌热休克蛋白GroEL1的研究进展[J]. 微生物与感染, 2010, 5(3): 186-191.
[3] Zeng S, Constant P, Yang D, et al. Cpn60.1 (GroEL1) contributes to mycobacterial crabtree effect: implications for biofilm formation[J]. Front Microbiol, 2019, 10: 1149.
[4] Esteban J, García-Coca M. Mycobacterium biofilms[J]. Front Microbiol, 2017, 8: 2651.
[5] Ansari MY, Batra SD, Ojha H, et al. A novel function of Mycobacterium tuberculosis chaperonin paralog GroEL1 in copper homeostasis[J]. FEBS Lett, 2020, 594(20): 3305-3323.
[6] Yang D, Klebl DP, Zeng S, et al. Interplays between copper and Mycobacterium tuberculosis GroEL1[J]. Metallomics, 2020, 12(8): 1267-1277.
[7] Canova MJ, Kremer L, Molle V. The Mycobacterium tuberculosis GroEL1 chaperone is a substrate of Ser/Thr protein kinases[J]. J Bacteriol, 2009, 191(8): 2876-2883.
[8] 许涛, 张丽, 钱中清, 等. H37Rv结核分枝杆菌蛋白脯氨酸-谷氨酸8(PE8)的表达及其兔多克隆抗体制备[J]. 细胞与分子免疫学杂志, 2020, 36(6): 549-554.
[9] Mobed A. DNA based vaccines against mycobacterium tuberculosis: recent progress in vaccine development and delivery system[J]. Iran J Immunol, 2020, 17(4): 255-274.
[10] Sergeeva M, Romanovskaya-Romanko E, Zabolotnyh N, et al. Mucosal influenza vector vaccine carrying TB10.4 and HspX antigens provides protection against mycobacterium tuberculosis in mice and guinea pigs[J]. Vaccines, 2021, 9(4): 394.
[11] Davenne T, Mcshane H. Why don't we have an effective tuberculosis vaccine yet[J]. Expert Rev Vaccines, 2016, 15(8): 1009-1013.
[12] Khawbung JL, Nath D, Chakraborty S. Drug resistant tuberculosis: a review[J]. Comp Immunol Microbiol Infect Dis, 2021, 74: 101574.
[13] Sielaff B, Lee KS, Tsai FTF. Structural and functional conservation of mycobacterium tuberculosis GroEL paralogs suggests that GroEL1 Is a chaperonin[J]. J Mol Biol, 2011, 405(3): 831-839.
[14] Hakiem OR, Parijat P, Tripathi P, et al. Mechanism of HrcA function in heat shock regulation in mycobacterium tuberculosis[J]. Biochimie, 2020, 168: 285-296.
[15] Trutneva KA, Shleeva MO, Demina GR, et al. One-year old dormant, non-culturable mycobacterium tuberculosis preserves significantly diverse protein profile[J]. Front Cell Infect Microbiol, 2020, 10: 26.
[16] Sevalkar RR, Arora D, Singh PR, et al. Functioning of mycobacterial heat shock repressors requires the master virulence regulator PhoP[J]. J Bacteriol, 2019, 201(12): e00013-e00019.
[17] 陈勇, 胡建国, 吕合作, 等. 可激活人γδT细胞的结核杆菌耐热多肽抗原的初步纯化与活性鉴定[J]. 免疫学杂志, 2002, 18(3): 176-179, 182.
[18] 刘玲, 李璟, 范蕾, 等. 新冠病毒Nsp1蛋白结构与功能的生物信息学分析及原核表达[J]. 中国人兽共患病学报, 2022, 38(7): 566-576.
[19] 徐本锦, 范蕾, 杜淼, 等. 新冠病毒核衣壳蛋白结构与功能的生物信息学分析及原核表达[J]. 中国免疫学杂志, 2022, 38(24): 2963-2972.
[20] 陈晓文, 陈越, 高婧华, 等. 鸟分枝杆菌MAV2928基因编码蛋白的生物信息学分析[J]. 中国病原生物学杂志, 2021, 16(1): 17-21.
[21] 郝锐, 刘仟仟, 王璐, 等. 乙肝病毒核心蛋白序列特征的生物信息学分析[J]. 中国病原生物学杂志, 2022, 17(1): 31-36, 42.
