肺炎链球菌TCSs中WalR的克隆表达、保守性及抗原表位分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.07.028

生物技术通报 ›› 2016, Vol. 32 ›› Issue (7): 194-199.doi: 10.13560/j.cnki.biotech.bull.1985.2016.07.028

肺炎链球菌TCSs中WalR的克隆表达、保守性及抗原表位分析

韩道宾¹, 骆诗露¹, 黄健², 朱杰华², 陈泽慧², 闵迅¹

1. 遵义医学院医学检验系,遵义 563000；
2. 遵义医学院附属医院检验科,遵义 563000

收稿日期:2015-12-02 出版日期:2016-07-25 发布日期:2016-07-25
作者简介:韩道宾,男,硕士研究生,研究方向：细菌致病分子机制研究;E-mail：you15865885832@126.com
基金资助:
国家自然科学基金资助项目（81460317）,贵州省科技攻关项目（黔科合SY字（2012）3092号）

Clone and Expression of WalR in Streptococcus pneumonia TCSs and Analyzation of Conservation and Antigenic Epitope

HAN Dao-bin¹, LUO Shi-lu¹, HUANG Jian², ZHU Jie-hua², CHEN Ze-hui², MIN Xun¹

1. Department of Medical Laboratory of Zunyi Medical University,Zunyi 563000;
2. Clinical Laboratory Department of Zunyi Medical University Affiliated Hospital,Zunyi 563000

Received:2015-12-02 Published:2016-07-25 Online:2016-07-25

摘要/Abstract

摘要： 通过构建PET28a-WalR表达载体并在大肠杆菌BL21中表达WalR重组蛋白,以评价其对C57小鼠的免疫原性;并分析其在不同血清型肺炎链球菌中的保守性和B细胞的抗原表位。以肺炎链球菌D39血清型WalR基因为模板,构建重组质粒PET28a-WalR并转入BL21诱导表达目的蛋白质。采用ClustalX 2.1软件对WalR蛋白在不同血清型S.pn中的保守性进行分析;利用DNASTAR软件对其B细胞抗原表位进行分析。结果显示,成功构建PET28a-WalR重组表达载体,经IPTG诱导后有大量高纯度的目的蛋白质,但其主要以包涵体形式存在;WalR蛋白在不同血清型肺炎链球菌中高达99.4%,其B细胞抗原表位可能位于9-16、35-42、95-111、113-135、150-161、200-218等氨基酸序列位点。成功获得大量以包涵体形式表达的肺炎链球菌WalR重组蛋白,并对其保守性和抗原表位进行了系统分析。

关键词: 肺炎链球菌, WalR, 重组蛋白, 保守性, 抗原性

Abstract: This work aims to express WalR protein in Escherichia coli BL21 through constructing recombinant expression vector PET28a-WalR and to analyze the immunogenicity in the experimental animal C57 mice,as well as to test the conservation in differentStreptococcus pneumonia serotypes and B cell antigenic epitopes. Using the WalR gene of S. pneumonia D39 as the template,the recombinant expression vector PET28a-WalR was constructed and then transferred into Escherichia coli BL21 for induced expressing target protein. The conservation of WalR protein in varied S. pneumonia serotypes was analyzed by ClustalX 2.1 software,and the B cell antigenic epitopes was analyzed by DNASTAR Lasergene v7.1. As result,recombinant expression vector PET28a-WalR was constructed successfully and much target protein was obtained by inducement of IPTG,however mainly in inclusion body. The conservation of WalR protein was up to 99.4% among different S. pneumonia serotypes,and the antigenic epitopes of WalR protein were likely located in 9-16,35-42,95-111,113-135,150-161,and 200-218 of amino acid sequence. In conclusion,recombinant S. pneumonia WalR protein in the form of inclusion body was successfully acquired,and the conservation and antigenic epitope were analyzed.

Key words: Streptococcus pneumonia, WalR, recombinant protein, conservation, antigenicity

韩道宾, 骆诗露, 黄健, 朱杰华, 陈泽慧, 闵迅. 肺炎链球菌TCSs中WalR的克隆表达、保守性及抗原表位分析[J]. 生物技术通报, 2016, 32(7): 194-199.

HAN Dao-bin, LUO Shi-lu, HUANG Jian, ZHU Jie-hua, CHEN Ze-hui, MIN Xun. Clone and Expression of WalR in Streptococcus pneumonia TCSs and Analyzation of Conservation and Antigenic Epitope[J]. Biotechnology Bulletin, 2016, 32(7): 194-199.

参考文献

[1] Sugita G, Hotomi M, Sugita R, et al. Genetic characteristics of Haemophilus influenzae and Streptococcus pneumoniae isolated from children with conjunctivitis-otitis media syndrome[J]. Journal of Infection and Chemotherapy：Official Journal of the Japan Society of Chemotherapy, 2014, 20（8）：493-497.
[2] Reijtman V, Gagetti P, Faccone D, et al. Macrolide resistance in Streptococcus pneumoniae isolated from Argentinian pediatric patients suffering from acute otitis media[J]. Revista Argentina Demicrobiología, 2013, 45（4）：262-266.
[3] Altinkanat GG, Soysal A, Kuzdan C, et al. Serotype distribution and antibiotic susceptibilities of Streptococcus pneumoniae causing acute exacerbations and pneumonia in children with chronic respiratory diseases[J]. Mikrobiyoloji Bülteni, 2013, 47（4）：684-692.
[4] Min X, Zhang X, Wang H, et al. Protection against pneumococcal infection elicited by immunization with glutamyl tRNA synthetase, polyamine transport protein D and sortase A[J]. Vaccine, 2012, 30（24）：3624-3633.
[5] DosSSR, Passadore LF, Takagi EH, et al. Serotype distribution of Streptococcus pneumoniae isolated from patients with invasive pneumococcal disease in Brazil before and after ten-pneumococcal conjugate vaccine implementation[J]. Vaccine, 2013, 31（51）：6150-6154.
[6] Huang WZ, Wang JJ, Chen HJ, et al. The heat-inducible essential response regulator WalR positively regulates transcription of sigI, mreBH and lytE in Bacillus subtilis under heat stress[J]. Research in Microbiology, 2013, 164（10）：998-1008.
[7] Liu P, Chen X, Huang Q, et al. The Role of CzcRS Two-component systems in the heavy metal resistance of Pseudomonas putida X4[J]. International Journal of Molecular Sciences, 2015, 16（8）：17005-17017.
[8] Dhiman A, Bhatnagar S, Kulshreshtha P, et al. Functional characterization of WalRK：A two-component signal transduction system from Bacillus anthracis[J]. FEBS Open Bio, 2014, 4：65-76.
[9] Gotoh Y, Doi A, Furuta E, et al. Novel antibacterial compounds specifically targeting the essential WalR response regulator[J]. The Journal of Antibiotics, 2010, 63（3）：127-134.
[10] 朱学喆, 赵志强, 谢贵林. 肺炎链球菌疫苗研制进展[J]. 微生物学免疫学进展, 2013, 41（1）：58-64.
[11] Chu CY, Liu CH, et al. Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant[J]. Vaccine, 2015, 33（1）：92-99.
[12] 闵迅, 黄美容, 黄健, 等. 肺炎链球菌疫苗候选蛋白质谷氨酰胺tRNA合成酶的保守性与抗原性分析[J]. 临床检验杂志, 2012, 30（6）：442-446.
[13] Audouy SA, van Selm S, van Roosmalen ML, et al. Development of lactococcal GEM-based pneumococcal vaccines[J]. Vaccine, 2007, 25（13）：2497-2506.
[14] Moraes JJ, Stipp RN, Harth-Chu EN, et al. Two-component system VicRK regulates functions associated with establishment of Streptococcus sanguinis in biofilms[J]. Infection and Immunity, 2014, 82（12）：4941-4951.
[15] Wayne KJ, Li S, Kazmierczak KM, et al. Involvement of WalK（VicK）phosphatase activity in setting WalR（VicR）response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells[J]. Molecular Microbiology, 2012, 86（3）：645-660.
[16] Zheng L, Yan M, Fan F, et al. The essential walK histidine kinase and walR regulator differentially mediate autolysis of RN4220[J]. Journal of Nature and Science, 2015, 1（6）：e111.
[17] Sun L, Andika IB, Shen J, et al. The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies[J]. Molecular Plant Pathology, 2014, 15（5）：466-478.
[18] Yamaguchi H, Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies[J]. Biomolecules, 2014, 4（1）：235-251.
[19] Pereira LM, Silva LR, Alves JF, et al. A simple strategy for the purification of native recombinant full-length human RPL10 protein from inclusion bodies[J]. Protein Expression and Purification, 2014, 101：115-120.

肺炎链球菌TCSs中WalR的克隆表达、保守性及抗原表位分析

Clone and Expression of WalR in Streptococcus pneumonia TCSs and Analyzation of Conservation and Antigenic Epitope

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