摘要
目的:构建苦豆子凝集素基因(SAL)原核和真核表达载体及制备高效价的抗血清。方法:将编码苦豆子凝集素基因(SAL)的ORF成熟肽片段分别克隆到原核表达载体pMAL-p2x和真核表达载体pcDNA3.1中;重组质粒pMAL-p2x-SAL转化大肠杆菌BL21(DE3)诱导表达。对pcDNA3.1-SAL质粒进行酶切和测序鉴定;采用"DNA prime-protein boost"策略免疫小鼠制备多克隆抗体;用ELISA和Western blot检测血清效价和特异性。结果:制备的多克隆抗体效价为1:102400;Western blot分析表明多克隆抗血清特异性较好。结论:苦豆子凝集素多克隆抗血清具有很好的特异性,为进一步研究苦豆子凝集素的生物学特性奠定了基础。
Objective：To construct the prokaryotic and eukaryotic expression vectors of Sophora alopecuroides lectin gene SAL and prepare a high titer polyclonal antibody against recombinant SAL protein. Method：The ORF sequence of SAL was cloned to pMAL -p2X and pcD-NA3. 1 vectors, respectively. The recombinant plasmid pMAL - p2x - SAL was then transformed into Escherichia coli BL21 （ DE3 ） and in-duced for expression. The recombinant plasmid pcDNA3.1 - SAL was characterized by enzymatic digestion and sequencing. Strategy of DNA prime - protein boost was employed to immunized mice to generate polyclonal antibodies. ELISA and Western blot assays were used to assess polyclonal antibodies features. Result：The titer of the polyclonal antibody against the recombinant SAL protein was about 1：102400 by indirect ELISA. Western blot analysis showed a favorable specificity of the polyclonal antibodies. Conclusion：The polyclonal antiserum to recombinant Sophora alopecuroides lectin exhibits a high titer and favorable specificity, which lays a foundation for further studying the biological property of Sophora alopecuroides lectin.
出处
《生物技术》
CAS
CSCD
北大核心
2013年第5期66-69,共4页
Biotechnology
基金
新疆维吾尔自治区自然科学基金项目("苦豆子凝集素的分子构象和作用对象的研究"
2010211A01)资助~~
关键词
苦豆子凝集素
克隆与表达
联合免疫
多克隆抗血清
Sophora alopecuroides lectin
Cloning and expression
Combined immunization
Polyclonal antiserum
