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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (8): 10-18    DOI: 10.13523/j.cb.20180802
研究报告     
真核表达猪白细胞介素17及其生物活性研究 *
邓定浩,肖永乐,唐健雪,杨鑫,高荣()
四川大学生命科学学院 生物资源与生态环境教育部重点实验室 动物疫病防控与食品安全四川省重点实验室 成都 610065
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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摘要：

目的:为真核表达猪白细胞介素17(IL-17),研究产物在细胞培养下的免疫生物活性。方法:通过PCR扩增出猪IL-17基因并插入到真核表达载体pVAX1,然后转染到IPEC-J2细胞、HaCaT细胞和L02细胞中。在转染后第24、48和72h收集细胞,第48h收集上清液。收集细胞通过实时荧光定量PCR检测相关免疫基因的表达水平,收集上清液通过抑菌试验检测相关抗菌肽的生物活性。结果:采用pVAX1载体构建了表达猪IL-17的重组质粒,转染到细胞中。证实IL-17基因能诱导抗菌肽基因(RegⅢ、S100A8和BD2)的表达,显著上调JAK-STAT信号通路基因(JAK1、STAT1和STAT3)和细胞因子基因(IL-6、IL-12和TNF-α)的表达。此外,细胞上清液能够在不同程度上抑制大肠杆菌及金黄色葡萄球菌的增殖。结论:成功将猪IL-17基因真核表达,其表达产物能诱导效应细胞表达多种细胞因子,产生多种抗菌肽,具有抑菌能力;这为进一步研发猪IL-17作为抗菌免疫分子制剂奠定了初步基础。
关键词： 猪;  白细胞介素17;  细胞转染;  免疫;  抑菌效应    
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
收稿日期: 2018-03-19 出版日期: 2018-09-11
ZTFLH:  Q786  
基金资助: 成都市科技攻关项目(2015NY-02-0028-NC);四川省科技项目(2017HH0023);四川省科技项目(2016RZ0034);科技部国际合作项目(2011DFA10101103)
通讯作者: 高荣     E-mail: gaorong96@163.com
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引用本文:

邓定浩,肖永乐,唐健雪,杨鑫,高荣. 真核表达猪白细胞介素17及其生物活性研究 *[J]. 中国生物工程杂志, 2018, 38(8): 10-18.

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.

链接本文:

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

基因 引物(5'-3') 退火温
度/℃
pPPIA-F AGACAGCAGAAAACTTCCGTG 52
pPPIA-R ACTTGCCACCAGTGCCATTA
hGAPDH-F GTCAGTGGTGGACCTGACCT 61
hGAPDH-R AGGGGAGATTCAGTGTGGTG
pIL-17-F CAGACGGCCCTCAGATTACTCCA 61
pIL-17-R AGCCCACTGTCACCATCACTTTCT
hIL-17-F ACCAATCCCAAAAGGTCCTC 50.4
hIL-17-R CACTTTGCCTCCCAGATCAC
pRegⅢγ-F CCACCGAGGGCTTGGAA 50.5
pRegⅢγ-R GCAACGTAATTGAGCACATCAGA
hRegⅢα-F TATGGCTCCCACTGCTATGCCT 51
hRegⅢα-R TCTTCACCAGGGAGGACACGAA
pS100A8-F GCGTAGATGGCGTGGTAA 50
pS100A8-R GCCCTGCATGTGCTTTGT
hS100A8-F ATGCCGTCTACAGGGATGAC 55.5
hS100A8-R ACGCCCATCTTTATCACCAG
pBD2-F CCAGAGGTCCGACCACTA 56.8
pBD2-R GGTCCCTTCAATCCTGTT
hBD2-F CATGAGGGTCTTGTATCTCCTCT 57.6
hBD2-R CCTCCTCATGGCTTTTTGCAGC
pJAK1-F TTTGAGAAGTCCGAGGTGCTA 60
pJAK1-R CAGGATCTGCTTCTTCAGGTG
hJAK1-F TTCTACATGGGGGGATAG 56
hJAK1-R TAAGTATGGAAACCCTCTAA
pSTAT1-F TCTGGCACAGTGGCTAGAAAATC 57.4
pSTAT1-R GAAAACGGATGGTGGCAAAC
hSTAT1-F AGTCTTGGCACCTAACGTGCTG 54
hSTAT1-R AGTTCGTACCACTGAGACATCCTG
基因 引物(5'-3') 退火温
度/℃
pSTAT3-F TGCAGCAGAAAGTGAGCTAC 57.2
pSTAT3-R CCGGTCTTGATGACTAATGG
hSTAT3-F GGAGGAGTTGCAGCAAAAG 56
hSTAT3-R TGTGTTTGTGCCCAGAATGT
pIL-6-F GGCAAAAGGGAAAGAATCCAG 52.5
pIL-6-R CGTTCTGTGACTGCAGCTTATCC
hIL-6-F ACTCACCTCTTCAGAACGAATTG 54.8
hIL-6-R CCATCTTTGGAAGGTTCAGGTTG
pIL-12-F TAGCCACGAATGAGAGTTGCC 52.5
pIL-12-R AGGCACAGGGTTGTCATAAAAGA
hIL-12-F CCACTCCAGACCCAGGAATG 51
hIL-12-R GACGGCCCTCAGCAGGT
pTNF-α-F CGACTCAGTGCCGAGATCAA 54.5
pTNF-α-R CCTGCCCAGATTCAGCAAAG
hTNF-α-F CCCAGGGACCTCTCTCTAATC 52.4
hTNF-α-R ATGGGCTACAGGCTTGTCACT
表1  荧光定量PCR特异性引物
图1  IL-17基因的扩增
图2  PV17双酶切产物
图3  细胞中IL-17基因表达量的变化
图4  细胞中抗菌肽基因表达量的变化
图5  细胞中JAK-STAT信号通路基因表达量的变化
图6  细胞中细胞因子基因表达量的变化
图7  上清液对大肠杆菌的抑菌效果
图8  上清液对金黄色葡萄球菌的抑菌效果
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