您的位置：山东大学 -> 科技期刊社 -> 《山东大学学报（医学版）》

山东大学学报（医学版） ›› 2017, Vol. 55 ›› Issue (3): 64-69.doi: 10.6040/j.issn.1671-7554.0.2016.104

• 基础医学 • 上一篇    下一篇

弓形虫ROP19蛋白生物信息学分析及真核表达载体的构建

周剑1,赵晗婷1,吕刚2,王琳3,李启航1,朱美艳1,王志林1,何深一2   

  1. 1.山东大学临床医学院, 山东 济南 250012;2.山东大学基础医学院病原生物学研究所, 山东 济南 250012;3.济南市儿童医院神经电生理科, 山东 济南 250022
  • 收稿日期:2016-02-02 出版日期:2017-03-10 发布日期:2017-03-10
  • 通讯作者: 王琳. E-mail:wanglinfenghuaxue@163.com何深一. E-mail:shenyihe@sdu.edu.cn E-mail:wanglinfenghuaxue@163.com
  • 基金资助:
    国家自然科学基金(81071373)

Bioinformatics analysis and construction of an eukaryocyte vector of ROP19 protein in Toxoplasma gondii

ZHOU Jian1, ZHAO Hanting1, LÜ Gang2, WANG Lin3, LI Qihang1, ZHU Meiyan1, WANG Zhilin1, HE Shenyi2   

  1. 1. School of Medicine, Shandong Univeristy, Jinan 250012, Shandong, China;
    2. Department of Parasitology, School of Basic Medical Sciences, Shandong Univeristy, Jinan 250012, Shandong, China;
    3. Department of Nerve Electrophysiology, Jinan Childrens Hospital, Jinan 250022, Shandong, China
  • Received:2016-02-02 Online:2017-03-10 Published:2017-03-10

PDF (PC)

331

摘要： 目的 构建刚地弓形虫PRU株ROP19蛋白的真核表达载体并检测其表达。 方法 利用生物信息学方法分析弓形虫ROP19蛋白的理化性质并比较弓形虫蛋白ROP19和SAG1的B细胞表位及T细胞表位,采用分子克隆技术构建重组真核表达质粒pEGFP-C1-ROP19(pROP19),经PCR扩增、酶切及测序鉴定正确后,体外转染HEK293T细胞,置于倒置荧光显微镜下观察荧光的表达情况。细胞转染质粒48 h后收集裂解细胞,提取蛋白后用Western blotting检测重组真核表达质粒pROP19在体外细胞中的表达。 结果 生物信息学分析显示ROP19主要位于膜上,具备比弓形虫SAG1更优异的抗原表位;RT-PCR结果表明重组真核载体pROP19成功构建,Western blotting结果显示ROP19蛋白可以被抗STAG小鼠血清识别。 结论 成功获得重组真核表达质粒pROP19,并能在真核细胞内表达。

关键词: ROP19蛋白, 真核表达, 刚地弓形虫, 生物信息学

Abstract: Objective To construct and express the eukaryotic expression vector of ROP19 protein of Toxoplasma gondii PRU strain. Methods The physical and chemical characteristics of ROP19 protein were analyzed, and the B cell epitopes and T cell epitopes of ROP19 were compared with SAG1 using bioinformatics. The recombinant eukaryotic expression plasmid pROP19 was constructed by the molecular cloning technology. After being identified by PCR, restriction enzyme cleavage and sequencing, pROP19 was transfected into the HEK293T cells. The cells from different groups(control, pEGFP-C1 and pROP19)were respectively detected with fluorescence microscope under blue laser. The ROP19 protein was detected by Western blotting. Results The ROP19 protein was mainly located in the membrane and had better antigenic index than SAG1. The plasmid was constructed successfully. Western blotting showed that the expressed proteins could be recognized by anti-STAg mouse sera. Conclusion The eukaryotic expression vector pROP19 is constructed and expressed in eukaryotic cells.

Key words: Eukaryotic expression, ROP19 protein, Toxoplasma gondii, Bioinformatics

中图分类号: 

  • R382.5
[1] Chen J, Xu MJ, Zhou DH, et al. Canine and feline parasitic zoonoses in China[J]. Parasit Vectors, 2012, 5: 152.
[2] Nardoni S, Angelici MC, Mugnaini L, et al. Prevalence of Toxoplasma gondii infection in Myocastor coypus in a protected Italian wetland[J]. Parasit Vectors, 2011, 4: 240.
[3] Tian YM, Dai FY, Huang SY, et al. First report of Toxoplasma gondii seroprevalence in peafowls in Yunnan Province, Southwestern China[J]. Parasit Vectors, 2012, 5: 205.
[4] Meng M, He S, Zhao G, et al. Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1(SAG1)and 14-3-3 protein in BALB/c mice[J]. Parasit Vectors, 2012, 5: 273.
[5] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans[J]. Int J Parasitol, 2000, 30(12-13): 1217-1258.
[6] Hoseinian KK, Ghaffarifar F, DSouza S, et al. Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis[J]. Vaccine, 2011, 29(4): 778-783.
[7] Alarcon JB, Waine GW, Mcmanus DP. DNA vaccines: technology and application as anti-parasite and anti-microbial agents[J]. Adv Parasitol, 1999, 42: 343-410.
[8] Liu KY, Zhang DB, Wei QK, et al. Biological role of surface Toxoplasma gondii antigen in development of vaccine[J]. World J Gastroenterol, 2006, 12(15): 2363-2368.
[9] Han Y, Zhou A, Lu G, et al. Protection via a ROM4 DNA vaccine and peptide against Toxoplasma gondii in BALB/c mice[J]. BMC Infect Dis, 2017, 17(1): 59.
[10] Peixoto L, Chen F, Harb OS, et al. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses[J]. Cell Host Microbe, 2010, 8(2): 208-218.
[11] Kravetz JD, Federman DG. Toxoplasmosis in pregnancy[J]. Am J Med, 2005, 118(3): 212-216.
[12] Dziadek B, Gatkowska J, Brzostek A, et al. Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis[J]. Vaccine, 2011, 29(4): 821-830.
[13] Bai Y, He S, Zhao G, et al. Toxoplasma gondii: bioinformatics analysis, cloning and expression of a novel protein TgIMP1[J]. Exp Parasitol, 2012, 132(4): 458-464.
[14] Bhopale GM. Development of a vaccine for toxoplasmosis: current status[J]. Microbes Infect, 2003, 5(5): 457-462.
[15] Romano P, Giugno R, Pulvirenti A. Tools and collaborative environments for bioinformatics research[J]. Brief Bioinform, 2011, 12(6): 549-561.
[16] Shaw MK, Roos DS, Tilney LG. Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii[J]. Microbes Infect, 2002, 4(2): 119-132.
[17] Mishima M, Xuan X, Nishikawa Y, et al. Construction of recombinant feline herpesvirus type 1 expressing Toxoplasma gondii surface antigen 1[J]. Mol Biochem Parasitol, 2001, 117(1): 103-106.
[18] Chen H, Chen G, Zheng H, et al. Induction of immune responses in mice by vaccination with Liposome-entrapped DNA complexes encoding Toxoplasma gondii SAG1 and ROP1 genes[J]. Chin Med J(Engl), 2003, 116(10): 1561-1566.
[19] 王琳,吕刚,韩亚莉,等. 弓形虫棒状体蛋白7真核表达载体的构建及其免疫保护作用[J]. 山东大学学报(医学版), 2015,53(8): 11-15. WANG Lin, LÜ Gang, HAN Yali, et al. Construction of an eukaryotic vector of rhoptry protein 7 from Toxoplasma gondii and its immunoprotective effect[J]. Journal of Shandong University(Health Science), 2015, 53(8): 11-15.
[20] Yuan ZG, Zhang XX, He XH, et al. Protective immunity induced by Toxoplasma gondii rhoptry protein 16 against toxoplasmosis in mice[J]. Clin Vaccine Immunol, 2011, 18(1): 119-124.
[21] Yuan ZG, Zhang XX, Lin RQ, et al. Protective effect against toxoplasmosis in mice induced by DNA immunization with gene encoding Toxoplasma gondii ROP18[J]. Vaccine, 2011, 29(38):6614-6619.
[22] El-Kady IM. T-cell immunity in human chronic toxoplasmosis[J]. J Egypt Soc Parasitol, 2011, 41(1): 17-28.
[23] Lee TY, Hsu JB, Chang WC, et al. A comprehensive resource for integrating and displaying protein post-translational modifications[J]. BMC Res Notes, 2009, 2: 111.
[24] Gao J, Faraggi E, Zhou Y, et al. BEST: improved prediction of B-cell epitopes from antigen sequences[J]. PLoS One, 2012, 7(6): e40104. doi: 10.1371/journal.pone.0040104.
[25] Van Regenmortel MH. What is a B-cell epitope?[J]. Methods Mol Biol, 2009, 524: 3-20.
[26] Reichmann G, Dlugonska H, Fischer HG. Characterization of TgROP9(p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone[J]. Mol Biochem Parasitol, 2002, 119(1): 43-54.
[1] 张秋萍,朱慧志,吕川,夏咏琪,张秀. 基于生物信息学分析鉴定哮喘潜在的关键自噬和铁死亡相关基因[J]. 山东大学学报 (医学版), 2026, 64(1): 74-87.
[2] 王静,刘晓菲,曾荣,许长娟,张锦涛,董亮. 基于机器学习算法鉴定哮喘的坏死性凋亡相关生物标志物[J]. 山东大学学报 (医学版), 2024, 62(7): 21-32.
[3] 景凯,徐志伟,李乐平. 基于GEO数据库探究结直肠肿瘤相关基因及其功能[J]. 山东大学学报 (医学版), 2024, 62(1): 21-30.
[4] 秦金金,曹辰媛,邢杰杰,安燕,黄煜湘. 子痫前期相关Siglec-6核心基因的预测及生物信息学分析[J]. 山东大学学报 (医学版), 2024, 62(1): 31-37.
[5] 陈映均,刘同刚. 综合生物信息学分析鉴定乙型肝炎病毒相关肝细胞癌中异常甲基化修饰的差异表达基因[J]. 山东大学学报 (医学版), 2023, 61(9): 101-117.
[6] 常晴,刘佳,曲爱林,杨咏梅. 利用数据库信息分析NAMPT与肝癌的病理特征和免疫浸润的关联性[J]. 山东大学学报 (医学版), 2023, 61(4): 26-36.
[7] 刘腾,马迎春. 基于生物信息库病例分析ECT2在子宫内膜癌中的表达及临床意义[J]. 山东大学学报 (医学版), 2022, 60(8): 63-71.
[8] 李雁儒,李娟,李培龙,杜鲁涛,王传新. 胰腺癌不同进展期血清外泌体蛋白质组学分析[J]. 山东大学学报 (医学版), 2022, 60(10): 33-41.
[9] 华芳,张薇薇,吕波,辛玮. 生物信息学分析骨关节炎滑膜炎症相关基因和分子途径[J]. 山东大学学报 (医学版), 2021, 59(3): 10-17.
[10] 李灿楦,陈洁. 基于生物信息学分析乙酰辅酶A酰基转移酶1在肾透明细胞癌中的表达及作用机制[J]. 山东大学学报 (医学版), 2021, 59(2): 26-33.
[11] 栗英林,宋道庆,徐忠华. 应用生物信息学方法分析肾透明细胞癌中FKBP11的表达[J]. 山东大学学报 (医学版), 2020, 1(9): 45-51.
[12] 田宝睿,张永超,韩晓阳,田颖颖,王传玺. 利用数据库预测基因与胶质母细胞瘤的关联[J]. 山东大学学报 (医学版), 2020, 58(6): 8-13.
[13] 朱爱国,马振敕,王健. 利用上皮源性卵巢癌预后的多基因信息建立预测预后模型[J]. 山东大学学报 (医学版), 2019, 57(10): 80-85.
[14] 王琳, 吕刚, 韩亚莉, 郭晶晶, 赵群力, 何深一. 弓形虫棒状体蛋白7真核表达载体的构建及其免疫保护作用[J]. 山东大学学报（医学版）, 2015, 53(8): 11-15.
[15] 袁首道, 王凤琴, 张娇, 赵蔚明, 陈健行. 宫颈腺癌细胞与宫颈鳞癌细胞蛋白表达差异的质谱分析[J]. 山东大学学报（医学版）, 2014, 52(8): 14-21.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
版权所有 © 2018 《山东大学学报(医学版)》编辑部　
地址:山东大学科技期刊社（济南市历城区山大南路27号山东大学中心校区明德楼B座721室） 电话: 0531-88366918 E-mail: xbyxb@sdu.edu.cn
本系统由北京玛格泰克科技发展有限公司设计开发