【摘要】 目的构建EB病毒基因的原核表达载体,并在大肠杆菌中进行表达。分析非糖基化的EB病毒包膜糖蛋白gp85N端截短片段的抗原性。方法采用基因工程技术,以B95-8细胞(美洲绒猴外周血B淋巴细胞经EBV转化后的细胞系)[1]培养上清为模板,用PCR扩增EB病毒的BXLF2基因N端片段。PCR产物经HindⅢ和XhoⅠ双酶切,插入原核表达载体pGEX-5T中,构建pGEX5T-85N重组表达质粒,并在大肠杆菌BL21中诱导表达gp85N蛋白。纯化表达的蛋白用Westernblot鉴定,并免疫BALB/c小鼠。结果序列分析表明,插入片段的序列与GenBank登录的参考序列完全一致。重组表达的蛋白的相对分子质量(Mr)约为45000,同预期的大小相符。以纯化的可溶性重组蛋白免疫BALB/c小鼠,经ELISA检测获得了高效价的抗血清,且抗gp85单克隆抗体(mAb)可识别所表达的gp85N抗原。Westernblot显示,该抗原可与小鼠免疫血清起特异性反应。结论表达并纯化的EB病毒截短的gp85N重组蛋白具有良好的抗原性,为下一步分析所产生抗体的生物学特性提供了条件。更多还原

【Abstract】 AIM: To construct a prokaryotic recombinant vector of Epstein-Barr virus (EBV) membrane protein gp85, to express the protein in E.coli and characterize the antigenicity of this non-glycosylated protein. METHODS: The BXLF2 gene coding 5′-terminal truncated of EBV gp85 was amplified from the EBV strain B95-8 cell line with specific primers. After identification by the restriction digestion with Hind III and Xho I, the PCR product was inserted into the prokaryotic expression plasmid pGEX-5T and confirmed by sequencing. The constructed prokaryotic expression vector pGEX5T-85N was transformed into the competent E.coli BL21. The expressed recombinant protein gp85N was purified by affinity chromatography, characterized by Western blot, and used immunize BALB/c mice. The titer of antisevum from the immunized mice was detected by ELISA. RESULTS: Sequencing analysis revealed that the obtained truncated BXLF2 gene was identical to that published in GenBank and successfully cloned into pGEX-5T. SDS-PAGE showed that the expressed recombinant protein was partially soluble with a relative molecular mass of 45000. ELISA results indicated that the expressed gp85N was recognized by that the anti-gp85 mAb and contiserum with high titer was obtained from the immunized mice. CONCLUSION: The obtained recombinant gp85N with an excellent antigenicity should provide preliminary data for characterization of the antibody produced by the immunized mice.更多还原