摘要
目的:构建EB病毒的原核表达载体,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EB病毒B95-8细胞培养上清为模板,PCR扩增EB病毒的BXLF2基因N端片断。PCR产物经HindⅢ和XhoⅠ双酶切,并插入原核表达载体pGEX-5T,构建pGEX5T-85N重组表达质粒,在大肠杆菌BL21中诱导表达gp85N蛋白。纯化表达的蛋白进行蛋白免疫印迹鉴定,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。重组表达的蛋白的分子量约为45kD,与预期大小相符。可溶性重组蛋白纯化后免疫BALB/C小鼠,经ELISA检测获得了高效价的多克隆抗体,且gp85单抗可识别所表达的gp85N抗原。Western blot结果显示,该抗原可与小鼠免疫血清起特异反应。结论:表达并纯化的EB病毒的截短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条件。
Objective： To construct a prokaryotic recombinant vector for Epstein- Barr virus （EBV） membrane protein gp85, express the protein in E.coli and characterize the antigenicity of this non- glucosylated protein. Method： The BXLF2 gene coding for 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 expresslce plasmid pGEX - 5T 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 with glutathione agarcse and used for immunizing mice for 3 times. Results： Sequencing analysis revealed that truncated BXLF2 gene amplified was identical to that of published and successfully doned into pGEX - 5T. SDS - PAGE profile showed that the expressed recombinant protein was partially soluble with a relative molecular weight of 45 000 Dalton and ELISA results indicated that the anti - gp85 mAb recognize the expressed gp85N. Conclusion： The results showed that the recombinant gp85N with an excellent anti- genicity should provide preliminary data for characterizing antibody it produced.
出处
《福州总医院学报》
2008年第1期22-24,共3页
Journal of Fuzhou General Hospital
