EB病毒gp85N端片段的原核表达与初步鉴定

Prokaryotic expression and characterization of N-terminal truncated glycoprotein gp85 of Epstein-Barr virus

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。

AIM： To construct a prokaryotic recombinant vector of Epstein-Barr virus （EBV） membrane protein gp85, to express the protein in E. coil and characterize the antigenicity of this non-glycosylated protein. METHODS： The BX- LF2 gene coding 5＇-terminal truncated of EBV gp85 was amplified from the EBV strain 1395-8 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-ST and confirmed by sequencing. The constructed prokaryotic expression vector pGEXST-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-ST. 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 pro- duced by the immunized mice.