尼罗罗非鱼CD2-like基因克隆及其胞外区表达研究

Cloning and Analysis of Extracellular Domain Expression of CD2-like Gene in Nile Tilapia Oreochromis niloticus

依据数据库中预测的尼罗罗非鱼白细胞分化抗原2(CD2)基因(GenBank登录号:XM_005460661.4)序列,采用PCR扩增体质量(150±50)g尼罗罗非鱼CD2分子同源类似物(CD2-like)基因编码区片段,然后以其胞外区构建原核表达质粒pGEX-6P1-CD2L,并进行原核表达及条件优化,最后进行复性纯化,为进一步研究该CD2-like分子在罗非鱼中的免疫功能奠定基础。研究结果表明,罗非鱼CD2-like(OnCD2-like)基因(GenBank登录号:MN296489)编码区全长1110bp,其中胞外区长558 bp。构建OnCD2-like胞外区的原核表达质粒pGEX-6P1-CD2L,将该重组质粒导入大肠杆菌BL21后,成功表达OnCD2-like胞外段重组蛋白。原核表达结果显示,目的蛋白分子质量为20ku,主要以包涵体形式存在,而该包涵体蛋白复性纯化最佳条件为:37℃下,0.2mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导6h,经梯度稀释复性并纯化后可成功获得目的重组蛋白。

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.