摘要
目的 原核表达、纯化HBcAg-VEGF抗原表位融合蛋白,并分析其免疫原性。方法 生物信息学方法预测鼠血管内皮生长因子（vascular endothelial growth factor,VEGF）的B细胞抗原表位,采用重叠延伸PCR（splicing by overlap extension PCR,SOE-PCR）法将VEGF抗原表位基因插入到HBcAg基因的免疫优势区内,合成HBcAg-VEGF基因序列,克隆至原核表达载体pET-32a中,构建重组表达质粒pET-32a-HBcAg-VEGF,转化大肠埃希菌BL21（DE3）,IPTG诱导表达。表达产物经羟基磷灰石CHT层析、Sephacryl S-400HR凝胶过滤层析纯化后,与Al（OH）3佐剂混合,分别于第0、7、14、21天经肌肉免疫BALB/c小鼠1次,第28天采血,ELISA法检测血清中抗VEGF抗原表位抗体,VEGF受体结合抑制试验筛选具有良好免疫原性的VEGF优势抗原表位。对筛选出的优势抗原表位融合蛋白进行表达及纯化条件的优化。结果 生物信息学软件预测了6个VEGF的B细胞抗原表位;6个重组表达质粒经菌落PCR及测序证实构建正确;表达的HBcAg-VEGF抗原表位融合蛋白相对分子质量为14400～20100,纯度均在85%以上,除HBcAg-VEGF3以单体形式存在、不形成颗粒外,其他融合蛋白均能形成VLP;各组HBcAg-VEGF融合蛋白免疫小鼠后,均刺激机体产生了针对VEGF抗原表位肽的特异性抗体,其中HBcAg-VEGF1组抗体水平最高,且抑制VEGF与VEGFR结合的能力最强;采用优化的条件表达、纯化的HBcAg-VEGF1抗原表位融合蛋白纯度达95%以上。结论 筛选得到的HBcAg-VEGF1抗原表位融合蛋白显示出较好的免疫原性,为进一步研究其抗肿瘤效应奠定了基础。
Objective To express hepatitis B core antigen （HBeAg）-vascular endothelial growth factor （VEGF） epitope fusion protein in prokaryotic cells and analyze its immunogenicity. Methods The B cell antigen epitopes of VEGF were predicated by bioinformatie method, of which the cDNAs were inserted into the immunodominant region of HBcAg gene by splicing by overlap extension PCR （SOE-PCR）. The synthetic HBcAg-VEGF gene sequences were cloned into prokaryotic expression vector pET-32a. The constructed recombinant plasmid pET-32a-HBeAg-VEGF was transformed to E. coli B121 （DE3） for expression under induction of IPTG. The expressed product was purified by CHT and Sephacryl S-100 HR chromatography and mixed with aluminium hydroxide adjuvant, with which BALB/c mice were immunized i. m. on days O, 7, 14 and 21, and determined for antibody against VEGF in sera by ELISA on day 28. The dominant VEGF epitopes with high immunogenieity was screened by VEGF receptor binding inhibition test, and the conditions for expression and purification of dominant HBeAg-VEGF epitope fusion protein were optimized. Results Six B cell antigen epitopes of VEGF were predicated by bioinformatic software. PCR and sequencing proved that the six recombinant expression plas- raids were constructed correctly. The relative molecular masses of expressed HBcAg-VEGF epitope fusion proteins ranged from 14 400 to 20 100, while the purities were more than 85%. The fusion proteins, except HBeAg-VEGF3, formed VLPs. All the fusion proteins induced specific antibodies against VEGF epitope peptide, among which HBcAg-VEGF1 induced the highest antibody level and showed the strongest inhibiting ability to binding of VEGF and VEGFR. The HBcAg-VEGF1 epitope fusion protein expressed and purified under the optimized conditions reached a purity of more than 95%. Conclusion The screened HBcAg-VEGF1 epitope fusion protein showed high immunogenicity, which laid a foundation of further study on its anti-tumor effect.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第9期1278-1284,共7页
Chinese Journal of Biologicals
关键词
血管内皮生长因子
乙型肝炎核心抗原
抗原表位
融合蛋白
原核细胞
基因表达
免疫原性
Vascular endothelial growth factor （VEGF）
Hepatitis B core antigen （HBcAg）
Antigen epitope
Fusionprotein
Prokaryotic cells
Gene expression
Immunogenicity
