中国兽医科学2018，Vol.48Issue(5)：566-573,8.DOI:10.16656/j.issn.1673-4696.2018.0072
微小隐孢子虫P23基因真核表达质粒在HeLa细胞中的表达及其免疫小鼠血清抗体效价的检测
Expression of eukaryotic recombinant expression plasmid of Cryptosporidium parvum P23 gene in HeLa cells and detection of sera antibody titers in immunized mice
摘要
Abstract
In order to observe the expression of eukaryotic recombinant expression plasmid of Cryptosporidium parvum P23 gene in HeLa cells and the sera antibody titers in immunized mice,specific primers of C. parvum P23 gene were designed. The P23 gene was amplified by PCR from genomic DNA of C. parvum oocysts,and the amplified product was inserted into pVAX-1. The identified recombinant plasmids pVAXl-P23 were transfected into HeLa cells and the expression of recombinant P23 protein was detected by indirect immunofluorescence assay and Western-blot. ICR mice were immunized with the recombinant plasmid pVAXl-P23 and the sera antibody titers were detected by indirect ELISA. The results showed that the P23 gene of C. parvum length with 336 bp was amplified successfully. The inserted sequence and insert position of gene P23 in the eukaryotic recombinant plasmid pVAXl-P23 were both correct. The indirect immunofluorescence assay and Western-blot analysis showed that the recombinant eukaryotic plasmid pVAXl-P23 was successfully expressed in HeLa cells. Significant antibody level was detected in mice from 2nd to 8th week post immunization. These results indicated that pVAXl-P23 was successfully constructed and expressed in HeLa cells,and pVAXl-P23 could induce significant humoral immune response in ICR mice. These results provide basis for next research of characteristics of C. parvum P23 and development of nucleic acid vaccine against cryptosporidiosis.关键词
微小隐孢子虫/P23基因/真核重组表达质粒/表达/抗体Key words
Cryptosporidium parvum/P23 gene/recombinant eukaryotic expression plasmid/expression/antibody分类
农业科技引用本文复制引用
游艳敏,陈兆国,米荣升,黄燕,杨衡,贾海燕,张烨华,张晓丽,刘辉,韩先干..微小隐孢子虫P23基因真核表达质粒在HeLa细胞中的表达及其免疫小鼠血清抗体效价的检测[J].中国兽医科学,2018,48(5):566-573,8.基金项目
上海市科技兴农重点攻关项目(沪农科攻字(2015)第1-10号) （沪农科攻字(2015）
2018年国家农产品质量安全风险评估项目(GJFP-201800703) （GJFP-201800703）
国家自然科学基金项目(31702025) （31702025）
宁夏回族自治区科技支撑计划项目(宁农科(2016)1号) （宁农科(2016）
中央级公益性科研院所基本科研业务费专项资金项目(2016JB13,2017JB03) （2016JB13,2017JB03）
