| 郝雪微 马翠 吴静红 李金桐 陈硕.人血清淀粉样蛋白SAA1 蛋白的原核重组表达[J].,2016,16(25):4829-4833 |
| 人血清淀粉样蛋白SAA1 蛋白的原核重组表达 |
| Recombination and Expression of SAA1 Fusion Protein |
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| DOI： |
| 中文关键词: 血清淀粉样蛋白 原核重组 冠心病 |
| 英文关键词: SAA1 Prokaryotic recombination Coronary heart disease |
| 基金项目:国家自然科学基金项目（81400353）；黑龙江省卫生厅科技项目（2013-120） |
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| 中文摘要: |
| 目的：重组及表达人血清淀粉样蛋白SAA1 融合蛋白，为进一步制备早期诊断冠心病的单克隆抗体做准备。方法：应用PCR
技术，以成人肝脏的cDNA 文库做模板，扩增出长度为315 bp 的人血清淀粉样蛋白SAA1 基因，并将其分别与带有HIS 标签的
PET-32a 载体及GST 标签的PGEX-4T-1 载体连接，转化入DH5琢感受态细胞中进行克隆并测序鉴定。并在大肠杆菌表达菌
BL21 菌中表达SAA1 融合蛋白，之后分别应用SDS-PAGE 及Western blot 技术检验重组蛋白的纯度。结果：经SDS-PAGE 凝胶
电泳后，我们分别看到了分子量为32kD 和分子量为38kD 的两个蛋白条带，这两个条带分别为PET- 32a- SAA1 重组蛋白和
PGEX- 4T-1- SAA1 重组蛋白，且这两种蛋白在超声后存在于菌液的沉淀中，证明在大肠杆菌中实现了不溶性的包涵体形式的表
达，经Western blot 鉴定，分别以HIS标签抗体和GST 标签抗体为第一抗体，以鼠二抗为第二抗体，证明所表达的蛋白质为SAA1
融合蛋白。结论：采用原核表达方法，经免疫纯化可获得高纯度的SAA1 融合蛋白，为进一步制备相应的单克隆抗体和开发以
SAA1 增高为指标之一的冠心病诊断试剂盒打下基础，为冠心病的诊断开辟新途径。 |
| 英文摘要: |
| Objective:To recombine and express a recombinant, Serum amyloidA (SAA1) fusion protein, in E. coli and further
prepare its monoclonal antibody which used for early diagnostic method of coronary artery heart disease.Methods:The gene encoding
SAA1 was cloned using PCR techniques from cDNA of Adult liver, the amplified gene was inserted into vectors pET-32a with HIS tag
and pGEX-4T-1 with GST tag, and was subcloned into DH-5α competent cells. The recombinant plasmids were constructed and
transformed into BL21 cells, and then the protein expression of SSA1 with His-Tag and GST-Tag expression (including His-Tag
and GST-Tag, respectively) were induced by IPTG and identified by SDS-PAGE and Western blotting.Results:A band of DNA about
315bp by DNA sequencing was confirmed as SAA1 gene, the expression systemwas constructed, and soluble expression of SAA1 fusion
protein was achieved successfully, which was identified by Western blotting.Conclusion:Fusion protein SAA1 with high purity could
be expressed in prokaryotic system. The study does a further preparation of its monoclonal antibody and lays a foundation for opening up
new ways for early diagnosis of coronary artery diseases. |
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