刚地弓形虫肌动蛋白profilin的原核表达和纯化

doi:10.13481/j.1671-587x.20170608

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (06): 1109-1114.doi: 10.13481/j.1671-587x.20170608

刚地弓形虫肌动蛋白profilin的原核表达和纯化

李会敏^1,2, 伊焕发¹

1. 吉林大学第一医院转化医学研究院移植免疫实验室, 吉林长春 130021;
2. 吉林大学第二医院检验科, 吉林长春 130041

收稿日期:2017-03-21 出版日期:2017-11-28 发布日期:2017-12-01
通讯作者: 伊焕发,教授,博士研究生导师(Tel:0431-88783042,E-mail:yihuanfa@jlu.edu.cn) E-mail:yihuanfa@jlu.edu.cn
作者简介:李会敏(1983-),女,吉林省长春市人,在读医学博士,主要从事免疫耐受与免疫调节方面的研究。
基金资助:
吉林省科技厅自然科学基金资助课题（20150101127JC）

Prokaryotic expression and purification of Toxoplasma gondii profilin protein

LI Huimin^1,2, YI Huanfa¹

1. Department of Transplant Immunology Laboratory, Institute of Translational Medicine, First Hospital, Jilin University, Changchun 130021, China;
2. Department of Clinical Laboratory, Second Hospital, Jilin University, Changchun 130041, China

Received:2017-03-21 Online:2017-11-28 Published:2017-12-01

摘要/Abstract

摘要： 目的：探讨刚地弓形虫肌动蛋白profilin （TgPRF）的原核表达体系和纯化条件，为后续的抗肿瘤免疫佐剂研究提供依据。方法：以弓形虫RH株速殖子的cDNA为模板，采用一对特定的引物扩增TgPRF基因的编码区。PCR产物双酶切后克隆入pET28a （+）载体中。重组的pET28a （+）-TgPRF质粒转化E.coli DH5α感受态细胞。双酶切鉴定阳性克隆，并选取测序正确的质粒转化E.coli BL21（DE3）表达菌，经IPTG诱导表达4 h，SDS-PAGE法检测TgPRF蛋白的表达，Western blotting法检测重组蛋白His-prolilin的表达。结果：PCR扩增产物长度为492 bp。经双酶切和测序，重组质粒pET28a-TgPRF连接产物构建成功。SDS-PAGE检测，目的蛋白在超声菌液的上清中表达。经Ni-NTA琼脂糖凝胶柱纯化，获得纯化的TgPRF蛋白（纯度>90%）。Western blotting检测，重组TgPRF蛋白能被Anti-His抗体识别。结论：成功构建重组质粒pET28a-TgPRF，并实现可溶性原核表达。

关键词: 刚地弓形虫, profilin, 蛋白纯化, 原核表达

Abstract: Objective:To discuss the prokaryotic expression system and purification conditions of Toxoplasma gondii profilin-like protein (TgPRF), and to provide basis for the study on anti-tumor immuno-adjuvant. Methods:The coding region of TgPRF gene was amplified with a pair of specific primers which were designed according to the cDNA oftachyzoites of Toxoplasma gondii RH strain. The PCR products were cloned into the pET-28a(+) vector after double enzyme digestion. The recombinant pET28a(+)-TgPRF plasmid was transformed into E.coli DH5α cells. The positive clones were selected by the double restrictive enzyme digestion and sequencing. The correct pET28a (+) -TgPRF plasmid was transformed into E.coli BL21(DE3) and induced for 4 h by IPTG. The expression of recombinant TgPRF protein was analyzed by SDS-PAGE method; the expression of recombinant protein His-profilin was detected by Western blotting method. Results:The length of product of PCR was 492 bp. The recombinant plasmid pET28a-TgPRF was confirmed by double restriction enzyme digestion and sequencing. The SDS-PAGE results showed that the target protein was expressed in E.coli BL21 (DE3) in bacteria supernatant. The purified TgPRF protein was obtained by Ni-NTA agarose gel column chromatography with the purity>90%. The Western blotting results revealed that the recombinant TgPRF protein could be recognized by Anti-His antibody. Conclusion:The recombinant plasmid pET28a-TgPRF is successfully constructed, and the TgPRF protein is obtained with the soluble prokaryotic expression.

Key words: Toxoplasma gondii, profilin, prokaryotic expression, protein purification

中图分类号:

R382.5

李会敏, 伊焕发. 刚地弓形虫肌动蛋白profilin的原核表达和纯化[J]. 吉林大学学报(医学版), 2017, 43(06): 1109-1114.

LI Huimin, YI Huanfa. Prokaryotic expression and purification of Toxoplasma gondii profilin protein[J]. Journal of Jilin University Medicine Edition, 2017, 43(06): 1109-1114.

参考文献

[1] Ovciarikova J, Lemgruber L, Stilger KL, et al. Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii[J]. Sci Rep, 2017, 7:42746. doi:10.1038/srep42746.
[2] Salazar Gonzalez RM, Shehata H, O'Connell MJ, et al. Toxoplasma gondii-derived profilin triggers human toll-like receptor 5-dependent cytokine production[J]. J Innate Immun, 2014, 6(5):685-694.
[3] Ge Y, Chen J, Qiu X, et al. Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii[J]. Int J Parasitol, 2014, 44(7):475-484.
[4] Krishnamurthy S, Konstantinou EK, Young LH, et al. The human immune response to Toxoplasma:Autophagy versus cell death[J]. PLoS Pathog, 2017, 13(3):e1006176.
[5] Tosh KW, Mittereder L, Bonne-Annee S, et al. The IL-12 response of primary human dendritic cells and monocytes to toxoplasma gondii is stimulated by phagocytosis of live parasites rather than host cell invasion[J]. J Immunol, 2016, 196(1):345-356.
[6] Che FY, Madrid-Aliste C, Burd B, et al. Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii[J]. Mol Cell Proteomics, 2011,10(1):1-14.
[7] Alam A, Bhatnagar RK, Relan U, et al. Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin, a multifunctional protein[J]. Mol Biochem Parasitol, 2013, 191(2):58-62.
[8] Koblansky AA, Jankovic D, Oh H, et al. Recognition of Profilin by Toll-like Receptor 12 is Critical for Host Resistance to Toxoplasma gondii[J]. Immunity, 2013, 38(1):119-130.
[9] Raetz M, Kibardin A, Sturge CR, et al. Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin[J]. J Immunol, 2013,191(9):4818-4827.
[10] Tanaka S, Kuroda Y, Ihara F, et al. Vaccination with profilin encapsulated in oligomannose-coated liposomes induces significant protective immunity against Toxoplasma gondii[J].Vaccine, 2014,32(16):1781-1785.
[11] Kramer K, Shields NJ, Poppe V, et al. Intracellular cleavable CpG oligodeoxynucleotide-antigen conjugate enhances anti-tumor immunity[J]. Mol Ther, 2017, 25(1):62-70.
[12] Hanagata N. CpG oligodeoxynucleotide nanomedicines for the prophylaxis or treatment of cancers, infectious diseases, and allergies[J].Int J Nanomedicine, 2017, 12:515-531.
[13] Srivastava AK, Yolcu ES, Dinc G, et al. SA-4-1BBL/MPL as a novel immune adjuvant platform to combat cancer[J].Oncoimmunology,2016, 5(1):e1064580.
[14] Pyo KH, Lee YW, Lim SM, et al. Immune adjuvant effect of a Toxoplasma gondii profilin-like protein in autologous whole-tumor-cell vaccination in mice[J]. Oncotarget, 2016,7(45):74107-74119.
[15] Halonen SK, Weiss LM. Toxoplasmosis[J].Handb Clin Neurol, 2013,114:125-145.
[16] Yarovinsky F. Innate immunity to Toxoplasma gondii infection[J]. Nat Rev Immunol, 2014, 14(2):109-121.
[17] Engel MA, Neurath MF. Anticancer properties of the IL-12 family--focus on colorectal cancer[J]. Curr Med Chem, 2010, 17(29):3303-3308.
[18] Rankin EB, Yu D, Jiang J, et al. An essential role of Th1 responses and interferon gamma in infection-mediated suppression of neoplastic growth[J]. Cancer Biol Ther, 2003, 2(6):687-693.

刚地弓形虫肌动蛋白profilin的原核表达和纯化

Prokaryotic expression and purification of Toxoplasma gondii profilin protein

RichHTML

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	邵敏, 王新颖, 刘星, 王燕, 周鹤峰, 葛正龙. hISO基因原核表达载体的构建及其在大肠杆菌中的表达及鉴定[J]. 吉林大学学报(医学版), 2016, 42(01): 59-63.
[2]	李松，杨立彬，张舒岩，金羽，陈秀华，李新，刘玲，李树蕾,李树红. 建鲤重组Cystatin蛋白的原核表达、鉴定及多克隆抗体制备[J]. 吉林大学学报(医学版), 2014, 40(01): 204-209.
[3]	苏雅拉图，张永胜，陈宇杰，胡小平，王丽颖，万敏，吴柒柱. 重组小鼠IFN-β原核表达载体的构建及包涵体溶解条件的筛选[J]. 吉林大学学报(医学版), 2013, 39(4): 841-846.
[4]	宋天一，李菁华，张哲，史红艳,孙延波 . 蜱传森林脑炎病毒非结构蛋白1原核表达载体的构建[J]. 吉林大学学报(医学版), 2013, 39(3): 620-624.
[5]	刘彤，李洋，李丹妮*，耿楠希，李丰. 人p21活化激酶6基因截短区域的GST标签原核表达质粒的构建及其重组蛋白的表达[J]. 吉林大学学报(医学版), 2013, 39(3): 437-440.
[6]	张士尧\|浦昀\|徐坤\|李金华\|原野\|鞠文\|李娟. 重组钩端螺旋体多抗原位点同源合成基因的表达及其抗原性鉴定[J]. J4, 2012, 38(3): 404-408.
[7]	杨南扬, 缪璇, 伍贤军, 刘金美, 辻一郎, 李晓萌. 类泛素化修饰蛋白SUMO3 /GST融合蛋白表达载体的构建及表达[J]. J4, 2011, 37(6): 1010-1014.
[8]	陈光\|吴铭\|王刚\|孙旸. 重组人胶原蛋白肽在大肠杆菌中的高效表达[J]. J4, 2011, 37(4): 656-660.
[9]	徐丽娟,温剑平,史红艳,孙延波 . 人乳头瘤病毒16型E7基因原核表达载体的构建及表达[J]. J4, 2011, 37(4): 661-664.
[10]	孙玲,刘慧颖,李淑红\|宫鹏涛\|张西臣. 弓形虫GRA1基因的克隆与原核表达[J]. J4, 2011, 37(4): 631-635.
[11]	张耀方, 万晓珊, 赵宏鑫, 邵明龙, 杨苹, 孔祥鑫, 刘敏, 李校堃, 王会岩. FGF21［Tyr²⁰］突变体的克隆、表达及纯化[J]. J4, 2010, 36(6): 1088-1093.
[12]	周静，马吉春，赵小霞，方芳，宋献美，柳忠辉，台桂香. 重组人IL2-MUC1融合蛋白在大肠杆菌中的表达及鉴定[J]. J4, 2008, 34(5): 763-766.
[13]	杨立彬，李树蕾，谭岩，许淑芬，汪军. rhTL1A基因原核表达载体的构建、表达与纯化[J]. J4, 2008, 34(3): 415-419.
[14]	徐波,郑永晨,费邵阳，高航. 大鼠血红素氧化酶 1的基因克隆和原核表达[J]. J4, 2007, 33(6): 982-985.
[15]	徐波,郑永晨,费邵阳. 人CDC2基因的克隆和原核表达及鉴定[J]. J4, 2007, 33(5): 834-836.