利用RT-PCR扩增拟南芥螯合肽合成酶（AtPCS1）基因全序列，与表达载体载体pET28a连接，构建成原核表达质粒pET28a-AtPCS1并测序。将该质粒转化大肠杆菌BL21，用0.75mM IPTG诱导融合蛋白AtPCS1-His的表达，通过浸提法分离纯化该融合蛋白。用纯化的融合蛋白免疫小鼠，ELISA法测定抗血清的效价可达1：2000，抗血清同纯化的融合蛋白及拟南芥总蛋白的western-blot结果表明，制备的抗血清能与重组融合蛋白和拟南芥提取物发生抗原抗体反应。
The full length of AtPCS1 gene was amplified by RT-PCR. After sequencing, the gene was cloned into the expression vector pET28a to contrast AtPCS1 expression plasmid pET28a-AtPCS1. The E.coli BL21 contained the expression plasmid was induced by 0.75mM IPTG and the protein was purified using SDS buffer. Mouse was immunized with the purified protein. The results of ELISA showed that the titer of the anti-serum was about 1:2000 and the western-blot analysis of the anti-serum with purified protein AtPCS1-His or the total protein of Arabidopsis thaliana show the polyclonal anti-AtPCS1-His serum has high activity and speciality.
吴亦亮,王鸣刚,张红星,陈亮. AtPCS1基因的克隆、原核表达及抗血清的制备[J]. 中国生物工程杂志, 2006, 26(0): 139-142.
. Cloning, Expression the Recombinant AtPCS1 and Preparation. China Biotechnology, 2006, 26(0): 139-142.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I0/139
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