PD0721单链抗体的体外原核表达条件优化及蛋白质鉴定

doi:10.3969/j.issn. 1673-1689.2021.07.005

首页 > 过刊浏览>2021年第40卷第7期 >42-49. DOI:10.3969/j.issn. 1673-1689.2021.07.005

PD0721单链抗体的体外原核表达条件优化及蛋白质鉴定
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                        10.3969/j.issn. 1673-1689.2021.07.005
                    
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Optimization and Identification of Prokaryotic Expression Conditions of PD0721 Single-Chain Antibody in vitro

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为了构建抗表皮生长因子受体Ⅲ型突变体（epidermal growth factor receptor variant type Ⅲ，EGFRvⅢ）的单链抗体PD0721的大肠杆菌（Escherichia coli）表达系统，作者研究了PD0721重组蛋白在大肠杆菌BL21中的最佳表达条件，并建立相应的重组蛋白质纯化方法。首先构建重组表达质粒PD0721-pET-22b（+）并转化至大肠杆菌BL21中，再利用SDS-PAGE研究不同诱导剂IPTG浓度、不同诱导温度、不同诱导时间和不同菌体浓度对PD0721重组蛋白表达的影响。利用镍柱亲和层析开发PD0721重组蛋白的纯化方法，并使用蛋白质印迹法以及N端测序法对纯化后的蛋白质进行鉴定。菌落PCR及琼脂糖凝胶电泳表明，PD0721-pET-22b（+）重组质粒及PD0721重组大肠杆菌BL21构建成功. PD0721重组蛋白在体外原核表达的最佳诱导剂浓度为0.6 μmol/L，最佳温度为15 ℃，最佳诱导时间为12 h，最佳菌体浓度OD₆₀₀约为0.6。经过Ni²⁺柱亲和层析后，当咪唑的浓度为150 mmol/L，可以得到纯度较高的PD0721重组蛋白。SDS-PAGE电泳和Western Blotting结果表明，重组蛋白质产物的相对分子质量约为31 000，与理论相对分子质量一致，蛋白质的N端测序也与设计序列一致。作者成功构建了抗EGFRvⅢ抗体PD0721重组蛋白的体外原核表达体系，优化了最佳表达条件，并提供了纯化重组PD0721蛋白的可行方法。

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This work aimed to construct the Escherichia coli expression system, obtain the optimum expression conditions and establish a robust purification method of single-chain antibody PD0721 against epidermal growth factor receptor variant type Ⅲ(EGFRvⅢ). The recombinant plasmid PD0721-pET-22(+) was constructed and transformed into Escherichia coli BL 21.The optimum concentration of isopropyl-β-D-thiogalactoside (IPTG), induction temperature,induction time and cell concentration (OD₆₀₀) of the PD0721 expression were investigated by SDS-PAGE method. Ni²⁺ affinity chromatography was employed to purify PD0721 protein. Western Blotting and N-terminal sequencing were used to identify PD0721 protein. Colony PCR and agarose gel electrophoresis confirmed the successful construction of PD0721-pET-22b(+) recombinant plasmid and PD0721 recombinant Escherichia coli BL 21. The optimum IPTG concentration, temperature, induction time and OD₆₀₀ of PD0721 expression were 0.6 μmol/L, 15 ℃, 12 h and 0.6. PD0721 protein of high purity could be obtained when the concentration of imidazole was 150 mmol/L during the Ni²⁺ affinity purification. SDS-PAGE and Western Blotting results showed that the molecular weight of the recombinant target protein was about 31 000, consistent with the theoretical molecular weight. N-terminal sequencing of the protein was also consistent with the theoretical sequence of PD0721. In summary, the in vitro prokaryotic expression system of anti-EGFRvⅢ antibody PD0721 was successfully constructed. The optimum expression conditions were obtained, and a feasible method for purifying PD0721 protein was established.

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张煜彬,叶路芬,吴忠秀,薛维娜,何彬,杨畅,李勇军,王永林,刘亭. PD0721单链抗体的体外原核表达条件优化及蛋白质鉴定[J].食品与生物技术学报,2021,40(7):42-49.

ZHANG Yubin, YE Lufen, WU Zhongxiu, XUE Weina, HE Bin, YANG Chang, LI Yongjun, WANG Yonglin, LIU Ting. Optimization and Identification of Prokaryotic Expression Conditions of PD0721 Single-Chain Antibody in vitro[J]. Journal of Food Science and Biotechnology,2021,40(7):42-49.

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在线发布日期: 2021-07-26
出版日期: 2021-07-25

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