| 摘要: |
| 为了研究PHB2 蛋白的生物学活性及后续抗体的制备, 作者以前期构建的pMD18-T-Lm-PHB2质粒为模板, PCR 扩增Lm-PHB2基因全长CDS 区, PCR 产物经Hind III 和EcoR I 双酶切后连接至表达载体pET-32a, 构建重组质粒pET-32a-Lm-PHB2。将鉴定正确的重组质粒转化入大肠杆菌(Escherichia coli)Rosetta Blue, 经IPTG 诱导表达后, 表达产物通过SDS-PAGE 和Western blotting 进行检测, 利用镍柱亲和层析纯化得到纯度较高的目的蛋白rLm-PHB2。结果表明, 经PCR、双酶切和测序鉴定, 所构建的重组质粒pET-32a-Lm-PHB2序列正确, 表达产物存在于裂解菌液的上清和沉淀中, 经SDS-PAGE 和Western blotting 证实在约47 ku 处有目的蛋白rLm-PHB2 的表达条带。本研究构建了重组七鳃鳗(Lampetra japonica)PHB2 蛋白的原核表达载体, 并大量表达和纯化目的蛋白, 为该蛋白后续的抗体制备及生物活性研究奠定了基础。 |
| 关键词: 海洋生物学 七鳃鳗(Lampetra japonica) PHB2 原核表达 蛋白纯化 |
| DOI：10.11759/hykx20140310001 |
| 分类号: |
| 基金项目:高等学校博士学科点专项科研基金资助项目(20102136120002);大连市科技基金资助项目(2010J21DW018) |
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| Prokaryotic expression and purification of recombined lamprey PHB2 protein |
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| Abstract: |
| To study the in vivo biological activities of lamprey PHB2 protein and prepare the antibody of PHB2 protein, herein, we aimed to construct the soluble prokaryotic expression vector of Lm-PHB2, and obtain adequate amount of purified rLm-PHB2 protein. The Lm-PHB2 gene was amplified from the previously constructed pMD18-T-Lm-PHB2 plasmid template. The PCR products were subjected to Hind III and EcoR I digestion and then linked to a soluble expression vector pET-32a. The identified recombinant plasmid pET-32a-Lm-PHB2 was transformed into Rosetta Blue, and IPTG induced expression of rLm-PHB2 was confirmed by SDS-PAGE and Western blotting assay. The rLm-PHB2 fusion protein was purified using His affinity chromatography purification system to get highly purified protein. The correct construction of recombinant plasmid pET-32a-Lm-PHB2 was confirmed by PCR, restriction enzyme digestion and gene sequencing identification. Expression products were verified to present in the supernatant of bacteria lysis, indicting the successful soluble expression of rLm-PHB2. The SDS-PAGE and Western blotting results showed that the molecular weight of rLm-PHB2 protein was about 47 ku, corresponding with the anticipant size. The pET-32a-Lm-PHB2 prokaryotic expression vector was constructed correctly, and the soluble rLm-PHB2 protein was successfully expressedsucceeded. Ultimately, the rLm-PHB2 protein with high purity was obtained after purification. |
| Key words: marine biology lamprey PHB2 prokaryotic expression protein purification |
