Eukaryotic Expression of Porcine IL-17 Gene and its Bioactivity

doi:10.13523/j.cb.20180802

China Biotechnology

2018, Vol. 38

Issue (8): 10-18 DOI: 10.13523/j.cb.20180802

Eukaryotic Expression of Porcine IL-17 Gene and its Bioactivity

Ding-hao DENG,Yong-le XIAO,Jian-xue TANG,Xing YANG,Rong GAO(

)

Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,Key Laboratory for Animal Disease Prevention and Food Safety of Sichuan Province,College of Life Sciences,Sichuan University,Chengdu 610064,China

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Abstract

Objective: To study porcine interleukin-17(IL-17) eukaryotic expression and their biologic activity. Methods: The IL-17 gene was amplified by PCR and cloned into eukaryotic expression vector pVAX1,named PV17. PV17 was transfected into IPEC-J2 cells,HaCaT cells and L02 cells,thereafter the cells were collected on hours 24,48 and 72,the supernatants were collected on hours 48. Related genes expression levels in cells were analysed by qRT-PCR,bioactivity of antimicrobial peptides in supernatants were analyzed by bacteriostatic test in vitro. Results: The eukaryotic expression plasmid of the porcine IL-17 gene was constructed and transfected into the three eukaryotic cells,and could expressed in target cells. The expression levels of antimicrobial peptide genes (RegⅢ,S100A8 and BD2),JAK-STAT signaling pathway genes (JAK1,STAT1 and STAT3) and cytokine genes (IL-6,IL-12 and TNF-α) were significantly up-regulated. Furthermore,the supernatants have marked bacteriostatic effect on E. coli and S. aureus. Conclusion: The recombinant plasmid of the porcine IL-17 gene was constructed and expressed in target cells,and the expressed products elicited the significant increases of cytokines and antibacterial peptides,which manifested obvious antibacterial activities against E. coli and S. aureus drug-resistant bacteria,and facilitated the further development of porcine IL-17 as the antibacterial reagent.

Key words： Porcine IL-17 Cell transfection Immunity Antibacterial effect

Received: 19 March 2018 Published: 11 September 2018

ZTFLH:

Q786

Corresponding Authors: Rong GAO E-mail: gaorong96@163.com

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	Ding-hao DENG
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	Jian-xue TANG
	Xing YANG
	Rong GAO

Cite this article:

Ding-hao DENG,Yong-le XIAO,Jian-xue TANG,Xing YANG,Rong GAO. Eukaryotic Expression of Porcine IL-17 Gene and its Bioactivity. China Biotechnology, 2018, 38(8): 10-18.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180802 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I8/10

Table 1 The primers for qRT-PCR

Fig.1 Amplification of IL-17 gene by PCR Lane M:100bp DNA Ladder; Lane 1:Negative Control; Lane 2:IL-17 Amplification Product

Fig.2 Restriction enzyme digestion products of PV17 Lane M:2K plus DNA Marker; Lane 1:PV17 digestion products

Fig.3 Expression change of IL-17 gene in cells (a) Expression change of IL-17 gene in IPEC-J2 cells (b) Expression change of IL-17 gene in HaCaT cells (c) Expression change of IL-17 gene in L02 cells Note:Different letters represent multiple comparisons,different letters mean significant differences,the same letters mean no significant differences. The followings are the same as here

Fig.4 Expression change of antimicrobial peptide genes in cells (a) (b) (c) Expression change of RegⅢ gene in IPEC-J2 cells,HaCaT cells and L02 cells (d) (e) (f) Expression change of S100A8 gene in IPEC-J2 cells,HaCaT cells and L02 cells (g) (h) (i) Expression change of BD2 gene in IPEC-J2 cells,HaCaT cells and L02 cells

Fig.5 Expression change of JAK-STAT signaling pathway genes in cells (a) (b) (c) Expression change of JAK1 gene in IPEC-J2 cells,HaCaT cells and L02 cells (d) (e) (f) Expression change of STAT1 gene in IPEC-J2 cells,HaCaT cells and L02 cells (g) (h) (i) Expression change of STAT3 gene in IPEC-J2 cells,HaCaT cells and L02 cells

Fig.6 Expression change of cytokine genes in cells (a) (b) (c) Expression change of IL-6 gene in IPEC-J2 cells,HaCaT cells and L02 cells (d) (e) (f) Expression change of IL-12 gene in IPEC-J2 cells,HaCaT cells and L02 cells (g) (h) (i) Expression change of TNF-α gene in IPEC-J2 cells,HaCaT cells and L02 cells

Fig.7 Inhibition effect of the supernatants on the proliferation of E. coil (a) (b) (c) Inhibition effect of the three supernatants on the proliferation of standard E. coil (d) (e) (f) Inhibition effect of the three supernatants on the proliferation of drug-resistant E. coil Note:PC means positive control,NC means negative control,BC means blank control. The followings are the same as here

Fig.8 Inhibition effect of the supernatants on the proliferation of S. aureus (a) (b) (c) Inhibition effect of the three supernatants on the proliferation of standard S. aureus (d) (e) (f) Inhibition effect of the three supernatants on the proliferation of drug-resistant S. aureus


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