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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (12): 14-20    DOI: 10.13523/j.cb.20181203
Orginal Article     
The Construction and Functional Analysis of Staphylococcal Enterotoxin-like K and GFP Fusion Protein
HE Ya-nan1,2,SUN Yu-liang1,2,REN Ya-kun2,LIANG Sheng-ying2,YANG Fen2,3,LIU Yan-li1,2,**(),LIN Jun-tang1,2,3
1 College of Life Science and Technology,Xinxiang Medical University, Xinxiang 453003,China
2 Henan Key Laboratory of Medical Tissue Regeneration, Xinxiang Medical University, Xinxiang 453003,China
3 College of Biomedical Engineering, Xinxiang Medical University, Xinxiang 453003,China
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Abstract  

Objective: The aim of this study is to construct the genetically engineered bacteria with full-length fusion gene of staphylococcal enterotoxin-like K (SElK) and green fluorescent protein (GFP), and further examine the biological activity of SElK-GFP fusion protein.Methods: SElK-GFP fusion gene was obtained by overlap PCR and cloned into the plasmid of pET28a. After being confirmed by colony PCR, double digestion and sequence, the successfully constructed pET28a-SElK-GFP was transformed into E.coli BL21, and the SElK-GFP was purified by Ni +-affinity magnetic beads. The proliferation of mouse derived spleen lymphocytes stimulated with SElK-GFP was examined by MTT assay; the concentration of IL-2 and IFN-γ in serum of the mice treated with SElK-GFP was examined by ELISA kits. Results: The SElK-GFP-producing strain was successfully constructed, and the green fluorescence can be observed in high purity SElK-GFP fusion protein. MTT assay showed that SElK-GFP could significantly stimulate the proliferation of spleen lymphocytes in a dose-dependent manner, and the concentration of IL-2 and IFN-γ in serum of the mice treated with SElK-GFP was significantly increased.Conclusion: SElK-GFP not only retained the green fluorescence signal of GFP, but also exhibited SElK superantgen activity, and provide a promising tool for the further study of the biological activity of SElK.

Key words： Staphylococcal enterotoxin-like K      Superantigen      T lymphocyte      Prokaryotic expression     
Received: 06 June 2018      Published: 10 January 2019
ZTFLH:  Q78  
Corresponding Authors: Yan-li LIU     E-mail: liuyanli198512@163.com
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Ya-nan HE
Yu-liang SUN
Ya-kun REN
Sheng-ying LIANG
Fen YANG
Yan-li LIU
Jun-tang LIN
Cite this article:

HE Ya-nan,SUN Yu-liang,REN Ya-kun,LIANG Sheng-ying,YANG Fen,LIU Yan-li,LIN Jun-tang. The Construction and Functional Analysis of Staphylococcal Enterotoxin-like K and GFP Fusion Protein. China Biotechnology, 2018, 38(12): 14-20.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181203     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I12/14

基因 引物 (5'- 3')
SEIK Sense CGG AAT TCC AAG GTG ATA TAG GAA
EcoR I
Antisense AGA GCC ACC TCC GCC TGA ACC GCC TCC ACC TAT CGT
The gene encoding linker peptide
TTC TTT ATA AGA
GFP Sense GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT ATG GTG
The gene encoding linker peptide
AGC AAG GGC GAG GA
Antisense TCG CTC GAG TTA CTT GTA CAG CTC GTC CAT GCC
Xho I Stop codon
SEIK-GFP Sense SEIK- Sense
Antisense GFP- Antisense
Table 1 The PCR primers of SElK, GFP and SElK-GFP gene
Fig.1 The construction and identification of pET-28a-SElK-GFP (1)Map of constructing SElK-GFP fusion gene by Overlap PCR (b) PCR products of SElK (lane 1 and 2), GFP (lane 3 and 4) and SElK-GFP fusion gene (lane 5 and 6) were analyzed by electrophoresis (c) The results of colony PCR (lane 1and 2) and double digestion of pET-28a-SElK-GFP by EcoR I and Xho I (lane 3 and 4)
Fig.2 Detection of green fluorescence during the expression of SElK-GFP (a) The expression of SElK-GFP induced with IPTG at 30℃ for 8h (b) Quantification of GFP fluorescence
Fig.3 The purification of SElK-GFP and the proliferation index of splenic lymphocytes stimulated by SElK-GFP in vitro (a) The expression of SElK-GFP induced with 1mmol/L IPTG at 30℃ for 8h. The samples were taken from 0h and 8h throughout the inducible expression period and were analyzed by the SDS-PAGE M: Protein molecular mass standards (b) Murine splenic lymphocytes were incubated with different concentrations of SElK, GFP and SElK-GFP for 48h, and the proliferation capacity was determined by MTT assay. The red dashed line indicated the PI of the BSA treated group (* P<0.05; ** P<0.01; NS: no significant difference)
Fig.4 Concentration of IL-2 and IFN-γ in serum of the mice treated with SElK-GFP (* P<0.05; ** P<0.01)
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