Efficient Secretory Expression of Optimized Mouse Interleukin-33 Gene in Mammalian Cells

doi:10.13523/j.cb.20190307

China Biotechnology

2019, Vol. 39

Issue (3): 46-55 DOI: 10.13523/j.cb.20190307

Efficient Secretory Expression of Optimized Mouse Interleukin-33 Gene in Mammalian Cells

Fu-lan GAO^1,²,Jia-long QI²,Cong-yan SHU²,Hang-hang XIE²,Wei-wei HUANG²,Cun-bao LIU²,Xu YANG²,Wen-jia SUN²,Hong-mei BAI²,Yan-bing MA^2,^**(

)

1 Kunming Medical University,Kunming 650500,China
2 Chinese Academy of Medical Science&Peking Union Medical College,Institute of Medical Biology,;

Download:

HTML

PDF(6242KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective: Interleukin (IL)-33 has important immunoregulatory effects and plays an important role in the disease. The aim of this paper is to achieve high-efficient expression of IL-33 in mammalian cells through gene optimization, and provide an important basis for disease mechanism research and vaccine immunoadjuvant application. Methods: The gene optimization for mammalian cell expression was carried out according to codon preference. The optimized and not optimized mIL-33 gene sequences were chemically synthesized. The human CD8αsignal peptide sequence was ligated to 5'end of the mIL-33 genes and led to fused CD8α+mIL-33 (Not optimized) and CD8α+mIL-33 (optimized) gene fragments by bridge PCR. The gene sequences of CD8α+ mIL-33 (Not optimized) or CD8α+mIL-33 (optimized) and EGFP expressing green fluorescent protein were respectively constructed into different expression units of plasmid pBudCE4.1 which has double expression units, and then 293FT cells were transfected with the recombinant plasmids using lipofectamine 3000 or PEI. The expression of recombinant proteins was detected by Western blot and ELISA. The expressed mIL-33s were used to stimulate macrophage Raw264.7, and the TNFα level of the culture supernatant was detected by ELISA to confirm the biological activity of expressed products. Results: The constructed recombinant plasmids were confirmed by restriction endonuclease digestion and sequencing analyses. The transfection efficiency of lipofectamine 3000 was higher than that of PEI. Western blot and ELISA showed higher levels of expression and secretion of IL-33 using optimized gene version. The expression level of mIL-33 in 293FT cells under the control of EF-1α promoter or CMV promoter was comparable. Expressed mIL-33 showed dose-dependent biological activity to stimulate TNF-α production in RAW264.7. Conclusion: The codon optimization significantly improved the secretory expression of mIL-33 with biological activity in mammalian cells, which laid a foundation for further research.

Key words： Key Laboratory of Infectious Disease Vaccine Research and Development Engineering Technology Research Center of Infectious Disease Vaccine Research and Development Kunming 650118 China)

Received: 22 August 2018 Published: 12 April 2019

ZTFLH:

Q291

Corresponding Authors: Yan-bing MA E-mail: may@imbcams.com.cn

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Fu-lan GAO
	Jia-long QI
	Cong-yan SHU
	Hang-hang XIE
	Wei-wei HUANG
	Cun-bao LIU
	Xu YANG
	Wen-jia SUN
	Hong-mei BAI
	Yan-bing MA

Cite this article:

Fu-lan GAO,Jia-long QI,Cong-yan SHU,Hang-hang XIE,Wei-wei HUANG,Cun-bao LIU,Xu YANG,Wen-jia SUN,Hong-mei BAI,Yan-bing MA. Efficient Secretory Expression of Optimized Mouse Interleukin-33 Gene in Mammalian Cells. China Biotechnology, 2019, 39(3): 46-55.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190307 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I3/46

Table1 Primers used for the amplification of CD8α+mIL-33 and EGFP gene sequences

Fig.1 The construction map of recombinant pBudCE4.1 co-expressing mIL-33 and EGFP

Fig.2 The construction map of recombinant pBudCE4.1 expressing mIL-33

Table 2 Comparison of optimized and not optimized IL-33 gene sequences

Fig.3 Codon quality class distribution of different gene versions of mIL-33 for expression in Mammalian cells

Table 3 Structure characteristics of mRNA transcribed from different mIL-33 gene versions

Fig.4 Identification of recombinant plasmid by restriction enzyme digestion (a) M: DNA marker DL10000; 1: Plasmid pBudCE4.1/CD8α+ mIL-33(Not optimized)(PCMV) digested by PstⅠ+ BamHⅠ; 2: Plasmid pBudCE4.1/CD8α+ mIL-33(optimized)(PCMV)digested by PstⅠ+ BamHⅠ (b) 1: Plasmid pBudCE4.1/CD8α+ mIL-33(optimized)(PEF-1α) digested by KpnⅠ+ Bgl Ⅱ (c) 1: Plasmid pBudCE4.1/CD8α+ mIL-33(Not optimized)(PCMV)/EGFP(PEF-1α) digested by KpnⅠ+ Bgl Ⅱ; 2: Plasmid pBudCE4.1/CD8α+ mIL-33( optimized)(PCMV)/EGFP(PEF-1α) digested by KpnⅠ+ Bgl Ⅱ

Fig.5 Observation of eukaryotic plasmids transfection into 293FT cells with fluorescence microscope A、B: pBudCE4.1/CD8α+ mIL-33/EGFP with Lipofectamine 3000 C、D: pBudCE4.1/CD8α+ mIL-33/EGFP with PEI; 1: Fluorescence fields; 2: Fluorescence fields and clear fields superposition

Fig.6 Detection of recombinant mIL-33 protein in 293FT cell with Western blot 1: pBudCE4.1 alone; 2: pBudCE4.1/CD8α+ mIL-33(Not optimized) (P_CMV)/EGFP;3: pBudCE4.1/CD8α+ mIL-33(optimized)(P_CMV)/EGFP; 4: pBudCE4.1/CD8α+ mIL-33(optimized)(PEF-1α)

Fig.7 Detection of the expression and secretion of recombinant mIL-33 protein in 293FT cell with ELISA (a) Cells transfected with the recombinant plasmids (b) Culture supernatants

Fig.8 Dose dependent production of TNF-α by Raw264.7 cells stimulated with mIL-33


[1]	Baekkevold E, Roussigene M, Yamanaka T , et al. Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules. The American Joural of Pathology, 2003,163(1):69-79. doi: 10.1016/S0002-9440(10)63631-0 pmid: 12819012

[2]	Schmitz J, Owyang A, Oldham E , et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity, 2005,23(5):479-490. doi: 10.1016/j.immuni.2005.09.015 pmid: 16286016

[3]	Villarreal D, Weiner D . Interleukin 33: A switch-hitting cytokine. Current Opinion Immunology, 2014,28C:102-106. doi: 10.1016/j.coi.2014.03.004 pmid: 24762410

[4]	Milovanovic M, Volarevic V, Radosavljevic G , et al. IL-33/ST2 axis in inflammation and immunopathology. Immunologic Research, 2012,52(1-2):89-99. doi: 10.1007/s12026-012-8283-9 pmid: 22392053

[5]	Lu B, Yang M, Wang Q . Interleukin-33 in tumorigenesis, tumor immune evasion, and cancer immunotherapy. Journal of Molecular Medicine, 2016,94(5):535-43. doi: 10.1007/s00109-016-1397-0 pmid: 26922618

[6]	Dominguez D, Ye C, Gang Z , et al. Exogenous IL-33 restores dendritic cell activation and maturation in established cancer. The Journal of Immunology, 2017,198(3):1365-1375. doi: 10.4049/jimmunol.1501399 pmid: 28011934

[7]	Lucarini V, Ziccheddu G, Macchia I , et al. IL-33 restricts tumor growth and inhibits pulmonary metastasis in melanoma-bearing mice through eosinophils. OncoImmunology, 2017,6(6):e1317420. doi: 10.1080/2162402X.2017.1317420 pmid: 28680750

[8]	Moussion C, Ortega N, Girard J . The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: A novel ‘Alarmin’. PLoS One, 2008,3(10):e3331. doi: 10.1371/journal.pone.0003331

[9]	Tordesillas L, Goswami R, Benede S , et al. Skin exposure promotes a Th2-dependent sensitization to peanut allergens. Journal of Clinical Investigation, 2014,124(11):4965-4975. doi: 10.1172/JCI75660

[10]	Villarreal D, Wise M, Walters J , et al. Alarmin IL-33 acts as an immunoad juvant to enhance antigen-specific tumor immunity. Cancer Research, 2014,74(6):1789-1800. doi: 10.1158/0008-5472.CAN-13-2729 pmid: 24448242

[11]	Gao X, Wang X, Yang Q , et al. Tumoral expression of IL-33 inhibits tumor growth and modifies the tumor microenvironment through CD8+T and NK cells. Journal of Immunology, 2015,194(1):438-445. doi: 10.4049/jimmunol.1401344

[12]	Bonilla W, Frohlich A, Senn K , et al. The alarmin interleukin-33 drives protective antiviral CD8(+) T cell responses. Science, 2012,335(6071):984-989. doi: 10.1126/science.1215418 pmid: 22323740

[13]	Kim H, Lee S . Optimizing the secondary structure of human papillomavirus type 16 L1 RNA enhances L1 protein expression in Saccharomyces cerevisiae. Journal of Biotechnology, 2010,150(1):31-36. doi: 10.1016/j.jbiotec.2010.07.032 pmid: 20696192

[14]	Bartoszewski R, Jablonsky M, Bartoszewska S , et al. A synonymous single nucleotide polymorphism in DeltaF508 CFTR alters the secondary structure of the mRNA and the expression of the mutant protein. Journal of Biological Chemistry, 2010,285(37):28741-28748. doi: 10.1074/jbc.M110.154575

[1]	JIA Zhen-wei. The Function of SIRT1 and Its Role in Regulating Follicular Development and Oocyte Maturation[J]. China Biotechnology, 2020, 40(10): 51-56.

[2]	ZHANG Xiao-mao,GUO Jing-han,HONG Jie-fang,LU Hai-yan,DING Juan-juan,ZOU Shao-lan,FAN Huan. *Evaluation of UPR Response in Yeast by Using UPRE-lac* Z as a Reporter Gene**[J]. China Biotechnology, 2020, 40(10): 1-9.

[3]	PENG Xiang-lei,WANG Ye,WANG Li-nan,SU Yan-bin,FU Yuan-hui,ZHENG Yan-peng,HE Jin-sheng. Single-Primer PCR for Site-Directed Mutagenesis[J]. China Biotechnology, 2020, 40(8): 19-23.

[4]	ZENG Xiang-Yi,PAN Jie. Progress on Autophagy Regulation of Browning of White Adipose Cells[J]. China Biotechnology, 2020, 40(6): 63-73.

[5]	ZHOU Qin,WANG Shuang,ZHANG Ting,LI Shan-gang,CHEN Yong-chang. *T158M Single Base Editing of MECP2* Gene in Murine and Rhesus Monekey’s Embryos**[J]. China Biotechnology, 2020, 40(6): 31-39.

[6]	ZHANG Ying,KONG Xiang-xi,HOU Lin,WANG Shu-kun,YUAN Zeng-qiang. Role and Mechanism of Ozanimod (RPC1063) in Oligodendrocyte Precursor Cell Differentiation[J]. China Biotechnology, 2020, 40(6): 10-19.

[7]	GUO Yang,WAN Ying-han,WANG Jue,GONG Hui,ZHOU Yu,CI Lei,WAN Zhi-peng,SUN Rui-lin,FEI Jian,SHEN Ru-ling. *Toll-like Receptor 4 (TLR4) Gene Knockout Mouse Model Construction and Preliminary Phenotypic Analysis*[J]. China Biotechnology, 2020, 40(6): 1-9.

[8]	TANG Min,WAN Qun,SUN Shi-lei,HU Jing,SUN Zi-jiu,FANG Yu-ting,ZHANG Yan. The Effects of Hsa-miR-5195-3p on the Proliferation, Migration and Invasion of Human Cervical Cancer SiHa Cells[J]. China Biotechnology, 2020, 40(4): 17-24.

[9]	LI Tong-tong,SONG Cai-ling,YANG Kai-yue,WANG Wen-jing,CHEN Hui-yu,LIU Ming. Preparation and Neutralization Activity of Anti-Canine Parvovirus VP2 Protein Single-chain Antibody[J]. China Biotechnology, 2020, 40(4): 10-16.

[10]	ZHOU Yan-xing,HAN Meng,LIU Na-nv,HUANG Wei-wei. *Identification and Functional Study of G-quadruplexes in SENP1* Promoter**[J]. China Biotechnology, 2020, 40(1-2): 92-101.

[11]	ZHU Yongzhao,TAO Jin,REN Meng-meng,XIONG Ran,HE Ya-qin,ZHOU Yu,LU Zhen-hui,DU Yong,YANG Zhi-hong. Autophagy Protects Against Apoptosis of Human Placental Mesenchymal Stem Cells of Fetal Origin Induced by Tumor Necrosis Fator-α[J]. China Biotechnology, 2019, 39(9): 62-67.

[12]	ZHANG Yan-ting,GUO Yu-feng,YANG Zhi-hong,YANG Shao-qi. The Expression and Clinical Significance of CD177 ⁺ Neutrophils in Patients with Ulcerative Colitis[J]. China Biotechnology, 2019, 39(9): 58-61.

[13]	RAN Long-rong,XUE Ning,WANG Jian-rong,DU Ying-jian,MAO Jin-ju,ZHOU Quan-you YANG Zai-lin. Prognostic Significance of Peripheral Blood Lymphocyte Subsets in Patients with Hematologic Malignancies[J]. China Biotechnology, 2019, 39(9): 50-57.

[14]	YUAN Xiao-ying,WANG Ya-zhe,SHI Wei-hua,CHANG Yan,HAO Le,HE Ling-ling,SHI Hong-xia,HUANG Xiao-jun,LIU Yan-rong. Methodological Study on Flow Detection of PNH Clone and Its Clinical Screening Significance[J]. China Biotechnology, 2019, 39(9): 33-40.

[15]	HE Ling-ling,LUO Ting-ting,CHANG Yan,WANG Ya-zhe,YUAN Xiao-ying,SHI Wei-hua,LAI Yue-yun,SHI Hong-xia,QIN Ya-zhen,HUANG Xiao-jun,LIU Yan-rong. Analysis on the Laboratory Examination Characteristics in 28 Patients with Acute Megakaryoblastic Leukemia[J]. China Biotechnology, 2019, 39(9): 2-10.

Viewed

Full text

Abstract

Cited

Shared

Discussed