摘要
为了研究不同方式构建的单链抗体-碱性磷酸酶融合蛋白原核表达后的生物活性差异,并分析其可溶性表达的影响因素,本研究通过基因工程操作构建含有不同类型的单链抗体-碱性磷酸酶融合基因的表达载体,优化原核表达条件并诱导表达,比较分析宿主菌、连接肽、抗体的单体或二聚体等因素对融合蛋白可溶性表达和生物活性的影响。结果显示不同方式构建的单链抗体-碱性磷酸酶融合蛋白的活性差异显著,在单链抗体和碱性磷酸酶之间添加连接肽能够获得更高活性的融合蛋白,而单链抗体二聚体与碱性磷酸酶的融合蛋白的活性明显降低。因此,单链抗体-碱性磷酸酶融合蛋白的可溶性表达和活性与单链抗体及其构建方式有关,而抗体自身的特性起着关键性作用。
To ana lyze the biological activity and solubility of prokaryotic expressed fusion proteins containing single-chain variable fragment(sc Fv) antibody and alkaline phosphatase(AP), different types of sc Fv-AP fusion expression vectors were constructed by genetic manipulation. Subsequently, the prokaryotic expression conditions were optimized and the fusion proteins were induced for expression in Escherichia coli. Then, the solubility and activity of fusion proteins was compared to analyze the influencing factors of prokaryotic expression, including host bacteria, linker, and monomeric and dimeric forms of sc Fv antibody. The results showed that the different types of the constructed sc Fv-AP fusions had significant discrepancies in solubility and activity. Simultaneously, a linker added between sc Fv antibody and AP could improve the activity of fusion protein, while the dimeric sc Fv antibody had a reverse influence on the biological activity. In conclusion, the soluble expression and activity of sc Fv-AP fusion proteins are directly correlated with the sc Fv antibodies and their construction types, while the antibody properties may be a critical influencing factor.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2015年第8期1798-1803,共6页
Genomics and Applied Biology
基金
贵州省科技项目(黔科合J字[2013]2058号和黔科合LH字[2014]7092号)
贵州省高校优秀科技创新人才支持计划(黔教合KY字[2014]244)
贵阳市科技计划项目(筑科合同[20141001]37号)
贵州医科大学博士基金(院基金合同字第[2013]11号)共同资助
关键词
单链抗体
碱性磷酸酶
原核表达
可溶性
免疫检测
Single-chain variable fragment
Alkaline phosphatase
Prokaryotic expression
Solubility
Immunological detection
