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目的制备高效价人乳头瘤病毒(HPV)16型次要结构蛋白(L2)特异性兔血清抗体,并对其效价及其与HPV16 L2蛋白结合的特异性进行鉴定。方法将重组质粒PGEX-4T-1-HPV16 L2转化至E.coliBL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,SDS-PAGE和Western blot鉴定融合蛋白的抗原性;经镍螯合亲和层析柱(Ni-NTA Agarose)纯化HPV16 L2全长融合蛋白,纯化蛋白用佐剂乳化后经日本大耳白家兔背部皮下多点免疫注射,间隔2周后加强免疫,共2次,并分别于免疫前和每次免疫后1周取血,采用Western blot检测血清抗体的特异性,采用ELISA法检测血清抗体水平及变化趋势。结果家兔用HPV16 L2融合蛋白全程免疫后均产生了抗HPV16 L2抗体,ELISA法检测最高滴度达1∶256 000;Western blot检测显示,该抗体能识别HPV16 L2蛋白。结论用原核HPV16 L2全长蛋白免疫家兔,可获得高效价的血清抗体,该抗体具有与HPV L2蛋白结合的特异性。
Objective To prepare high titer human papillomavirus (HPV) 16 secondary structural protein (L2) specific rabbit serum antibody and identify its potency and its specificity to HPV16 L2 protein binding. Methods The recombinant plasmid PGEX-4T-1-HPV16 L2 was transformed into E.coli BL21 (DE3) and induced by isopropyl-β-D-thiogalactopyranoside (IPTG) The purified HPV16 L2 fusion protein was purified by Ni-NTA Agarose. The purified protein was emulsified with adjuvant and then subcutaneously injected into the back of Japanese white rabbits. Two weeks later, the immunizations were boosted twice in total. Blood was taken before immunization and one week after each immunization. The specificity of serum antibody was detected by Western blot. The serum antibody level and trend were detected by ELISA. Results All rabbits were immunized with HPV16 L2 fusion protein, and the highest titer of anti-HPV16 L2 antibody was obtained by ELISA. The highest titer of anti-HPV16 L2 antibody was 1:256 000 by Western blot. The result showed that HPV16 L2 protein was recognized by Western blot. Conclusion The prokaryotic HPV16 L2 full-length protein is used to immunize rabbits to obtain high-titer serum antibody which has the specificity of binding to HPV L2 protein.