EB病毒潜伏膜蛋白2B表位特异性兔血清抗体的制备和鉴定

Preparation and identification of rabbit serum polyclonal antibody specific to B-cell epitopes of recombinant latent membrane protein 2 (LMP2) of the Epstein-Barr virus

目的制备EB病毒(EBV)潜伏膜蛋白2(LMP2)串联B表位特异的兔血清多克隆抗体。方法利用分子克隆技术,将已鉴定的EBV LMP2蛋白的3个B细胞表位,即RIEDPPFNSLL,TLNLT和KSLSSTEFIPN,以柔性肽(GS)加以串联连接,经原核密码子优化后全基因合成,并经BamHⅠ和HindⅢ酶切位点克隆至pET32a(+)载体,测序鉴定正确的重组质粒转化至大肠埃希菌BL21(DE3),用异丙基硫代-B-D-硫代吡喃半乳糖苷(IPTG)进行诱导表达,对表达蛋白进行SDS-PAGE分析和Western blot鉴定。用镍螯合亲和层析柱(Ni-NTA Agarose)纯化表达产物,与佐剂乳化后皮下多点注射日本大耳白兔,隔周免疫1次,共3次。分别于免疫的0、2、4、6、8周取血,采用ELISA方法检测IgG抗体水平,采用Western blot和细胞免疫荧光法检测兔血清抗体的免疫反应性和结合特异性。结果 EBV LMP2B表位重组蛋白可通过原核表达系统进行表达,纯化的表达蛋白免疫兔可产生特异性IgG抗体,效价达1∶30 000。经Western blot法和细胞免疫荧光法鉴定,制备的兔血清抗体可识别LMP2重组蛋白和LMP2天然蛋白。结论成功获得了EBV LMP2串联B表位重组蛋白,制备的兔多克隆血清抗体效价高,特异性强,为LMP2的生物学和免疫学研究奠定了基础。

Objective To prepare rabbit serum polyclonal antibody specific to three B-cell linear epitopes of latent membrane protein 2（LMP2）of the Epstein-Barr virus（EBV）. Methods A bioinformatics approach was used to screen EBV-LMP2 for three B-cell epitopes（aa199-209（RIEDPPFNSLL）,318-322（TLNLT）,and 381-391（KSLSSTEFIPN））as potential immunodominant B-cell epitopes.The epitopes were joined with a flexible peptide linker（GS）,codons were optimized,and a gene encoding apeptide with multiple epitopes was synthesized.The gene was digested with BamHI/HindIII and then inserted into pET32a（＋）.The construct was verified through sequencing.Escherichia coli（DE3）was transformed with a pET32a（＋）/EBV LMP2multi-epitope plasmid and expression of the multi-epitope fusion protein was induced with IPTG and verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）and Western blot analysis.The fusion proteins were purified using Ni-NTA agarose and used to subcutaneously immunize rabbits with Freund＇s adjuvant once every other week,for 3times in total.Serum samples were collected from the immunized rabbits at week 0,2,4,and 6,and the levels of EBV LMP2-specific IgG antibodies were detected with indirect ELISA.The specific immune response and binding of the rabbit polyclonal antibody to EBV LMP2（the target antigen）were further verified with Western blot analysis and immunofluorescence analysis. ResultsThe recombinant plasmid was verified through sequencing,and recombinant EBV LMP2 with the B-cell epitopes of interest was expressed in a prokaryotic expression system.SDS-PAGE analysis indicated that recombinant EBV LMP2 had a molecular weight of 22 ku in the host E.coli BL21（DE3）,which is consistent with the predicted molecular weight.Rabbits immunized with the purified protein produced a specific IgG antibody response,and the titer of the specific IgG was as high as 1：30 000.Western blot analysis and immunofluorescence analysis indicated that the polyclonal antibody specifically recognized recombinant EBV LMP2 and native LMP2 was well. Conclusion Recombinant EBV LMP2 was successfully prepared with three B-cell epitopes,and polyclonal antibody prepared from immunized rabbits had a high titer and high specificity for the recombinant protein.These findings will facilitate further research on the biological and immunological function of EBV LMP2.