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| Cloning and Analysis of Extracellular Domain Expression of CD2-like Gene in Nile Tilapia Oreochromis niloticus |
| CHEN Funuan1, GAO Xiaolin1, ZENG Xiaolian1, XIE Caixia1, WANG Zhiwen1,2, LU Yishan1,2, WU Zaohe1, WANG Zhongliang1,2, WANG Bei1,2 |
1. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institute, Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;
2. Shenzhen Institute of Guangdong Ocean University, Shenzhen 518000, China |
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Abstract In this study, cluster of differentiation 2 (CD2) molecular homologous analogues (CD2-like) was cloned and identified in Nile tilapia Oreochromis niloticus with body weight of (150±50) g, and extracellular proteins of the molecule were obtained to provide a foundation for further research on the immune function of CD2-like molecule in Nile tilapia. Based on NCBI (Genbank: XM_005460661.4) predicted Nile tilapia CD2-like(OnCD2-like) gene sequences, OnCD2-like fragments were amplified by PCR. The prokaryotic expression plasmid pGEX-6P1-CD2L was constructed from its extracellular domain and the expression conditions were optimized to protein renaturation and purification. The openning reading frame (ORF) of OnCD2-like gene(GenBank: MN296489)was found to be 1110 bp and its extracellular domain was 558 bp in length. The extracellular domain molecular weight of target protein was about 20 ku, which was consistent with the expected results. The prokaryotic expression plasmid pGEX-6P1-CD2L of OnCD2-like gene extracellular domain gene was successfully constructed, and then the recombinant plasmid was introduced into BL21 and the OnCD2-like recombinant protein was successfully expressed, indicating that prokaryotic expression showed that the target protein was mainly in the form of inclusion bodies, and that the optimal expression of the inclusion body protein was observed under conditions of the induction of 0.2 mmol/L IPTG for 6 h at 37 ℃, and that finally the recombinant protein was successfully obtained after gradient dilution renaturation and purification.
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Received: 27 February 2020
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