重组人白细胞介素-27的表达、纯化及初步鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2026-0250

摘要/Abstract

摘要：

目的重组人白细胞介素-27（rh IL-27）单链融合蛋白在大肠杆菌中异源表达时易形成包涵体。通过引入促溶标签筛选与分子伴侣共表达的协同策略，以实现该蛋白的高比例可溶表达、纯化与初步鉴定。方法将人工合成的人IL-27单链融合基因克隆至pET-32a(+)载体。通过系统筛选6种促溶标签（XXA、SUMO、GST、NusA、TrxA及MBP）及5套分子伴侣共表达系统，以获得高可溶性表达方案。经镍柱亲和层析纯化目的蛋白，采用SDS-PAGE和Western blot进行鉴定。去除内毒素后，利用ELISA和CCK-8法检测rh XXA-IL-27与抗体的结合能力及其对人乳腺癌MCF-7细胞增殖的抑制作用。结果构建了IL-27融合基因及6种促溶标签表达载体。经筛选，XXA标签的促溶效果最佳，其融合蛋白的可溶性表达水平达64.91%。Western blot结果确认其为特异性目的蛋白。进一步引入分子伴侣pGro7与XXA标签共表达后，rh XXA-IL-27的最高表达量达46.4 mg/L，经镍柱纯化后获得较高浓度和纯度的目的蛋白。体外活性实验表明，30 μg/mL的rh XXA-IL-27处理48 h可显著抑制MCF-7细胞增殖。结论通过促溶标签（特别是XXA标签）与分子伴侣pGro7的协同策略，攻克了单链融合蛋白IL-27在大肠杆菌中易形成包涵体的难题，实现了XXA-IL-27融合蛋白的高效可溶性表达与纯化，获得了具有抑制MCF-7细胞增殖活性的XXA-IL-27。

关键词: 人白细胞介素-27, 原核表达, 促溶标签, 分子伴侣, MCF-7细胞, 细胞增殖抑制

Abstract:

Objective Recombinant human interleukin-27 (rh IL-27) single-chain fusion protein tends to form inclusion bodies when heterologously expressed in Escherichia coli. In this study, we aimed to achieve high-ratio soluble expression, purification, and preliminary characterization of the protein by employing a synergistic strategy that combined the screening of solubilization tags with the co-expression of molecular chaperones. Method The synthesized human IL-27 single-chain fusion gene was cloned into the pET-32a(+) vector. High-solubility expression schemes were obtained by systematically screening six solubilization tags (XXA, SUMO, GST, NusA, TrxA, and MBP) and five sets of molecular chaperone co-expression systems. The target protein was purified via nickel affinity chromatography and identified using SDS-PAGE and Western blot. After endotoxin removal, ELISA and CCK-8 assays were used to detect the binding affinity of rh XXA-IL-27 with antibodies and its inhibitory effect on the proliferation of human breast cancer MCF-7 cells. Result Expression vectors for the IL-27 fusion gene and six solubilization tags were constructed. Screening results indicated that the XXA tag provided the best solubilization effect, with the soluble expression level of its fusion protein reaching 64.91%. Western blot analysis confirmed the specificity of the target protein. Upon further introduction of the molecular chaperone pGro7 for co-expression with the XXA tag, the maximum expression of rh XXA-IL-27 reached 46.4 mg/L, and the target protein was obtained at high concentration and purity after nickel column purification. In vitro activity assays demonstrated that treatment with 30 μg/mL rh XXA-IL-27 for 48 h significantly inhibited the proliferation of MCF-7 cells. Conclusion The synergistic strategy combining solubilization tags (especially the XXA tag) with the molecular chaperone pGro7 successfully addressed the challenge of inclusion body formation for the single-chain fusion protein IL-27 expressed in E. coli. This approach realized the efficient soluble expression and purification of the XXA-IL-27 fusion protein, yielding XXA-IL-27 with potent inhibitory activity against MCF-7 cell proliferation.

Key words: human interleukin-27, prokaryotic expression, solubility-enhancing tags, molecular chaperones, MCF-7 cells, cell proliferation inhibition

任彦宵, 温赛, 谭天伟. 重组人白细胞介素-27的表达、纯化及初步鉴定[J]. 生物技术通报, 2026, 42(11): 318-327.

REN Yan-xiao, WEN Sai, TAN Tian-wei. Expression， Purification and Preliminary Identification of Recombinant Human Interleukin-27[J]. Biotechnology Bulletin, 2026, 42(11): 318-327.

图/表 13

图1 pET-32a-IL-27的表达载体构建示意图

Fig. 1 Schematic diagram of vector construction for pET-32a-IL-27

图2 不同标签表达载体构建示意图

Fig. 2 Schematic diagram of constructing vector with different tags

表 1 各质粒编码的伴侣蛋白质种类及诱导物

Table 1 Types of chaperone proteins encoded by each plasmid and inducers

No.	质粒 Plasmid	伴侣蛋白质 Chaperone protein	启动子 Promoter	诱导物 Inducer	抗性标记 Selection marker
1	pG-KJE8	dnaK-dnaJ-grpE groES-groEL	araB Pzt-1	L-Arabinose Tetracycline	Cm
2	pGro7	groES-groEL	araB	L-Arabinose	Cm
3	pKJE7	dnaK-dnaJ-grpE	araB	L-Arabinose	Cm
4	pG-Tf2	groES-groEL-tig	Pzt-1	Tetracycline	Cm
5	pTf16	tig	araB	L-Arabinose	Cm

图3 不同蛋白表达形式的SDS-PAGE电泳分析1：pET-32a（+）未诱导；2：pET-32a（+）全细胞；3：pET-32a（+）胞内上清；4：pET-32a（+）胞内沉淀（约20 kD处条带为空载体表达的标签多肽）；5：IL-27未诱导；6：IL-27全细胞；7：IL-27胞内上清；8：IL-27胞内沉淀；M：protein marker

Fig. 3 SDS-PAGE electrophoresis analysis of various protein expression form1: pET-32a (+) uninduced; 2: pET-32a (+) whole cells; 3: pET-32a (+) intracellular supernatant; 4: pET-32a (+) intracellular pellet (the band at approximately 20 kD corresponds to the tag polypeptide expressed by the empty vector); 5: IL-27 uninduced; 6: IL-27 whole cells; 7: IL-27 intracellular supernatant; 8: IL-27 intracellular pellet; M: protein marker

图4 SDS-PAGE电泳分析不同促溶标签对IL-27可溶性表达的影响M：Protein marker；1：IL-27胞内沉淀；2：IL-27胞内上清（54 kD）；3：XXA-IL-27胞内沉淀；4：XXA-IL-27胞内上清（71 kD）；5：SUMO-IL-27胞内沉淀；6：SUMO-IL-27胞内上清（66 kD）；7：MBP-IL-27胞内沉淀；8：MBP-IL-27胞内上清（91 kD）；9：GST-IL-27胞内沉淀；10：GST-IL-27胞内上清（76 kD）；11：NusA-IL-27胞内沉淀；12：NusA-IL-27胞内上清（105 kD）；13：TrxA-IL-27胞内沉淀；14：TrxA-IL-27胞内上清（66 kD）

Fig. 4 SDS-PAGE analysis of the effects of different solubility tags on the soluble expression of rh IL-27M: Protein marker; 1: IL-27 intracellular pellet; 2: IL-27 intracellular supernatant (54 kD); 3: XXA-IL-27 intracellular pellet; 4: XXA-IL-27 intracellular supernatant (71 kD); 5: SUMO-IL-27 intracellular pellet; 6: SUMO-IL-27 intracellular supernatant (66 kD); 7: MBP-IL-27 intracellular pellet; 8: MBP-IL-27 intracellular supernatant (91 kD); 9: GST-IL-27 intracellular pellet; 10: GST-IL-27 intracellular supernatant (76 kD); 11: NusA-IL-27 intracellular pellet; 12: NusA-IL-27 intracellular supernatant (105 kD); 13: TrxA-IL-27 intracellular pellet; 14: TrxA-IL-27 intracellular supernatant (66 kD)

图5 不同促溶标签下rh IL-27的可溶性灰度分析（A）与总表达量ELISA分析（B）不同字母表示组间差异显著（P<0.05），下同

Fig. 5 Densitometric analysis of rh IL-27 solubility (A) and ELISA quantification of total expression levels (B) with different solubility-enhancing tagsDifferent letters indicate significant differences among groups (P<0.05). The same below

图6 Western blot鉴定表达产物A：抗His标签抗体；B：抗IL-27 多克隆抗体。1：XXA-IL-27（71 kD）；2：SUMO-IL-27（66 kD）；3：GST-IL-27 （76 kD）；4：NusA-IL-27（105 kD）；5：TrxA-IL-27（66 kD）；6：MBP-IL-27（91 kD）；7：IL-27（54 kD）；M：protein marker

Fig. 6 Western blot identification of expressed productsA: Anti-His-tag antibody; B: anti-IL-27 polyclonal antibody; 1: XXA-IL-27 (71 kD); 2: SUMO-IL-27 (66 kD); 3: GST-IL-27 (76 kD); 4: NusA-IL-27 (105 kD); 5: TrxA-IL-27 (66 kD); 6: MBP-IL-27 (91 kD); 7: IL-27 (54 kD); M: protein marker

图7 SDS-PAGE分析分子伴侣共表达对XXA-IL-27可溶性的影响M：Protein marker；1：XXA-IL-27（pKJE7）胞内沉淀；2：XXA-IL-27（pTf16）胞内沉淀；3：XXA-IL-27（pGro7）胞内沉淀；4：XXA-IL-27（pG-KJE8）胞内沉淀；5：XXA-IL-27（pG-Tf2）胞内沉淀；6：XXA-IL-27 （pKJE7）胞内上清；7：XXA-IL-27 （pTf16）胞内上清；8：XXA-IL-27 （pGro7）胞内上清；9：XXA-IL-27 （pG-KJE8）胞内上清；10：XXA-IL-27 （pG-Tf2）胞内上清

Fig. 7 SDS-PAGE analysis of the effects of chaperone co-expression on XXA-IL-27 solubilityM: Protein marker; 1: XXA-IL-27 (pKJE7) intracellular pellet; 2: XXA-IL-27 (pTf16) intracellular pellet; 3: XXA-IL-27 (pGro7) intracellular pellet; 4: XXA-IL-27 (pG-KJE8) intracellular pellet; 5: XXA-IL-27 (pG-Tf2) intracellular pellet; 6: XXA-IL-27 (pKJE7) intracellular supernatant; 7: XXA-IL-27 (pTf16) intracellular supernatant; 8: XXA-IL-27 (pGro7) intracellular supernatant; 9: XXA-IL-27 (pG-KJE8) intracellular supernatant; 10: XXA-IL-27 (pG-Tf2) intracellular supernatant

图8 不同分子伴侣与XXA-IL-27共表达时的可溶性灰度分析（A）与总表达量ELISA分析（B）

Fig. 8 Densitometric analysis of XXA-IL-27 solubility (A) and ELISA quantification of total expression levels (B) upon co-expressed with different chaperones

图9 rh XXA-IL-27纯化过程的SDS-PAGE分析M：Protein marker；1：流穿液；2：20 mmol/L咪唑洗脱液；3：30 mmol/L咪唑洗脱液；4：75 mmol/L咪唑洗脱液；5：150 mmol/L咪唑洗脱液；6：300 mmol/L咪唑洗脱液；7：500 mmol/L咪唑洗脱液（未浓缩洗脱原液）

Fig. 9 SDS-PAGE analysis of rh XXA-IL-27 purificationM: Protein marker; 1: flow-through; 2: 20 mmol/L imidazole; 3: 30 mmol/L imidazole; 4: 75 mmol/L imidazole; 5: 150 mmol/L imidazole; 6: 300 mmol/L imidazole; 7: 500 mmol/L imidazole (unconcentrated eluate)

图10 TEV 酶切处理的 SDS-PAGE（A）与Western blot（B）分析M： Protein marker；（A） 1： TEV酶； 2： XXA-IL-27酶切前； 3： XXA-IL-27酶切后；（B） 1： XXA-IL-27酶切前； 2： XXA-IL-27酶切后

Fig. 10 SDS-PAGE (A) and Western blot (B) analysis of TEV cleavageM: Protein marker; (A) 1: TEV protease; 2: XXA-IL-27 before cleavage; 3: XXA-IL-27 after cleavage; (B) 1: XXA-IL before cleavage; 2: XXA-IL after cleavage

图11 XXA-IL-27 处理 48 h 对MCF-7 细胞形态的影响A：对照组；B：5 μg/mL处理组；C：30 μg/mL处理组

Fig. 11 Effect of XXA-IL-27 treatment for 48 h on the morphology of MCF-7 cellsA: Control group; B: 5 μg/mL treatment group; C: 30 μg/mL treatment group

图12 XXA-IL-27对MCF-7细胞存活率的影响及IC50测定A：细胞存活率柱状图；B：剂量反应及IC50拟合曲线

Fig. 12 Effect of XXA-IL-27 on MCF-7 cell viability and corresponding IC50 assessmentA: Bar chart of cell viability; B: dose-response profile and IC50 fitting curve

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