Epstein-Barr病毒包膜糖蛋白gp85的原核表达

目的:利用大肠杆菌BL21诱导表达GST-gp85融合蛋白,进行动物免疫制备多克隆抗体。方法:通过PCR反应从EB病毒转染的绒猴淋巴细胞系B95-8细胞中获得了gp85BXLF2基因,将此基因克隆到大肠杆菌表达载体pGEX-5T,得到阳性克隆pGEX5T-85。转化大肠杆菌BL21,IPTG诱导表达重组蛋白,表达产物经SDS-PAGE分析、尿素变性、复性和亲和层析纯化,并用初步纯化的蛋白免疫BALB/c小鼠。结果:SDS-PAGE可见在相对分子质量约100000处有蛋白条带,Western印迹表明该蛋白可与免疫的BALB/c小鼠血清及鼻咽癌血清起特异性反应。结论:在大肠杆菌细胞中成功表达了GST-gp85融合蛋白,该蛋白具有较好的抗原性和免疫原性。

Prokaryotic Expression of Glycoprotein gp85 of Epstein-Barr Virus

Objective: To express the GST-gp85 fusion protein using E.coli BL21 and prepare polyclonal antibody to the protein. Methods: The BXLF2 gene coding for 5'-terminal truncated of Epstein-Barr virus(EBV) gp85 was amplified from the B95-8 cell line transfected by EBV with specific primers. The PCR product was inserted into the prokaryotic expression plasmid pGEX-5T and confirmed by sequencing. The constructed prokaryotic expression vector, pGEX5T-85 was transformed into the competent E.coli BL21. The inclusion bodies were isolated and solubilized with urea and then washed, denatured and refolded. SDS-PAGE profile showed that the expressed recombinant protein was partially soluble with a relative molecular weight of 100 000 Dalton. Results: The expressed recombinant protein gp85 could initiate specific immunol reaction in mice. Conclusion: The recombinant gp85 show an excellent antigenicity and immunogenicity.