摘要
目的构建肾癌G250抗原肽与HBcAg重组基因的原核表达质粒pET28a(+)/C-G250肽-C,并通过大肠杆菌进行原核表达并纯化,分析表达融合蛋白的免疫原性及抗原性。方法 PCR扩增编码G250抗原中249～268氨基酸的基因片段,将G250肽插入去除主要免疫优势区的重组质粒pGEMEX/HBcAg中,获得重组质粒pGEMEX/C-G250肽-C,为更换酶切位点,以pGEMEX/C-G250肽-C为模板,PCR扩增C-G250肽-C基因片段,最终将目的基因亚克隆入原核表达质粒pET28a(+)中,IPTG诱导融合蛋白表达,利用Ni 2+-NTA亲和层析法纯化重组融合蛋白,SDSPAGE鉴定融合蛋白的表达,纯化后融合蛋白经腹腔注射免疫BALB/c小鼠,间接ELISA法检测小鼠血清中抗体的滴度。结果酶切及基因测序鉴定证实融合基因正确重组于表达质粒pET28a(+)中,原核表达并纯化后,该分离纯化融合蛋白纯度达95%以上,其相对分子质量约为22.35。BALB/c小鼠经4次免疫后,所有小鼠血清中均可检测到抗体,免疫第6周其血清中抗体滴度可达到1∶5.12×105,抗G250肽抗体滴度达到1∶6.4×104,抗HBcAg抗体滴度不超过1∶4×103。结论成功构建肾癌G250抗原肽与HBcAg重组基因原核表达质粒pET28a(+)/C-G250肽-C,在大肠杆菌中可高效表达,纯化后融合蛋白具有较强的免疫原性,能诱导小鼠产生高滴度和高特异性的抗体。
Objective To construct the G250 antigen peptide and HBcAg recombinant gene prokaryotic expression plasmid pET28a（＋）/C-G250 peptide-C and analyze the immunogenicity of the fusion protein expressed in E. coli BL21（DE3）. Methods The gene fragments of encoding G250 antigenic peptide/249-268 were amplified by PCR, the G250 peptide was inserted to remove the major immunodominant region of the recombinant plasmid pGEMEX / HBcAg to obtain the recombinant plasmid pGEMEX/ C-G250 peptide-C. For the replacement of the enzyme digestion sites, pGEMEX/C-G250 peptide-C was used as a template, C-G250 peptide-C gene fragment was amplified by PCR, and finally the target gene was subcloned into the prokaryotic expression plasmid pET28a （＋）. 1PTG was used to induce the expression of the fusion protein, which was then purified by NiZ＋-NTA affinity chromatography. SDS- PAGE was used to identify the protein expression. BALB/c mice were intraperitoneally injected with purified fusion protein and the serum antibody titer was detected by indirect ELISA. Results The recombinant gene was confirmed to be correct by the restriction enzyme digestion and DNA sequencing.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2014年第1期6-11,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
山东省自然科学基金资助项目(No.Y2006C04)~~
