摘要
目的构建无乳链球菌(group B Streptococcus agalactea,GBS)菌毛岛屿辅助蛋白BP、AP2主要抗原表位域融合表达重组质粒,进行高效表达,并分析其免疫活性。方法利用DNAstar生物信息软件Protean功能筛选BP和AP2主要抗原表位域,并引入15位柔性多肽,通过重叠延伸PCR技术扩增融合BP、AP2主要抗原表位域基因,PCR扩增串联体基因片段,克隆至原核表达载体p ET-30a(+),构建重组表达质粒p ET-30a(+)/BP+AP2,转化至大肠埃希菌BL21(DE3),IPTG诱导表达。表达的融合蛋白BP+AP2经亲和层析纯化后,进行Western blot鉴定,并免疫昆明小白鼠,检测其免疫原性。结果 PCR扩增出675 bp的BP和759 bp的AP2主要抗原域,经重叠延伸PCR获得了1 380 bp的BP、AP2串联体基因片段,DNA测序结果表明,无碱基的缺失、突变和移码。构建的重组表达质粒经诱导后获得表达,目的蛋白相对分子质量约55 000,主要以包涵体形式存在,纯化后纯度达96%以上,浓度为2.05 mg/ml。纯化的融合蛋白能被兔抗GBS阳性血清所识别,且具有良好的免疫原性。结论构建了GBS BP、AP2主要抗原表位域串联体重组表达质粒p ET-30a(+)/BP+AP2,表达产物具有良好的免疫活性,为进一步研究基于BP、AP2蛋白的致病机制、诊断制剂及基因工程疫苗奠定了基础。
Objective To construct the fusion expression vector for the major antigenic domain of accessory proteins BP and AP2 in pilus island of Streptococcus agalactiae and analyze the immune activity of expressed product. Methods The major antigenic domains of BP and AP2 were screened by DNAstar Protean software, in which 15 flexible peptides were introduced, and fused by overlapping extension PCR technique. The tandem gene fragment was amplified and cloned into vector p ET-30 a（＋）. The constructed recombinant plasmid p ET-30 a（＋）/BP ＋ AP2 was transformed to E. coli BL21（DE3）and induced with IPTG. The expressed fusion protein was purified by affinity chromatography and identified by Western blot, of which the immunogenicity was evaluated by immunization of Kunming mice. Results The major antigenic domains of BP and AP2, at lengths of 675 and 759 bp respectively, were amplified by PCR, based on which a tandem gene fragment at a length of 1 380 bp was obtained by overlapping extension PCR, which showed no base deletion, mutation or shift by DNA sequencing. The target protein, with a relative molecular mass of about 55 000, mainly existed i n a form of inclusion body, and reached a purity of more than 96% and a concentration of 2. 05 mg/ml after purification. The purified fusion protein was recognized by rabbit anti-GBS positive serum, which showed good immunogenicity. Conclusion Recombinant plasmid p ET-30 a（＋）/BP ＋ AP2 was constructed, and the expressed product showed high immune activity, which laid a foundation of further study on the pathogenic mechanisms, diagnostic kit and genetic engineering vaccines based on BP and AP2 proteins.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第11期1140-1145,1149,共7页
Chinese Journal of Biologicals
基金
国家自然科学基金项目(31560689)
内蒙古自治区乳源性致病菌防控工程技术研究中心开放课题(MDK2016037
MDK2016036
MDK2017016)
内蒙古自治区科技厅计划项目(2017)
内蒙古自治区"草原英才"创新团队项目(第七批)
关键词
无乳链球菌
菌毛岛屿
BP
AP2
串联表达
免疫活性
Streptococcus agalactiae
Pilus island
BP
AP2
Tandem expression
Immune activity
