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Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein
CHEN Qiu-li,YANG Li-chao,LI Hui,WEN Sha,LI Gang,HE Min
China Biotechnology ›› 2020, Vol. 40 ›› Issue (3) : 31-37.
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Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}Objectives: To express human Nek2 protein by prokaryotic expression system, optimize expression conditions and purify Nek2 protein and prepare anti-Nek2 polyclonal antibody.Methods: The fragment of Nek2 gene was constructed on the prokaryotic expression vector pET30a(+) and transformed into E. coli BL21 (DE3).Expression of the recombinant protein was induced with IPTG and the conditions such as induction temperature, IPTG inducer concentration and induction time were optimized.The protein was purified by staining with 250mmol/L KCl after 12% SDS-PAGE, and the purified Nek2 protein was identified by mass spectrometry.Polyclonal antibody was prepared by immunizing BALB/c mice with purified Nek2 protein, and the titer and specificity of polyclonal antibody was detected by ELISA, Western blot and immunofluorescence assays.Results: The recombinant prokaryotic expression plasmid pET30a(+)-Nek2 was constructed and the recombinant human Nek2 protein was mainly expressed in the form of inclusion bodies.Optimal induction conditions were 28℃,180r/min,IPTG concentration 0.2mmol/L and 32h induction for expression of recombinant protein.The purified protein by mass spectrometry was Nek2 protein, and the concentration of the purified Nek2 protein was 1.35mg/ml.The purified Nek2 protein was used to immunize mice. The titer of polyclonal antibody was greater than 1:243 000 and the polyclonal antibody exhibited a perfect antigenic specificity.Immunofluorescence assay showed that Nek2 was mainly localized in the cytoplasm and nucleus.Conclusion: The recombinant Nek2 protein was used to obtain a anti-Nek2 polyclonal antibody with perfect antigenic specificity.
Nek2 / Prokaryotic expression / Polyclonal antibody / KCl stain {{custom_keyword}} /
Fig.2 Recombinant human Nek2 protein expression and optimization of induction conditions(a)Expression of recombinant human Nek2 protein M:Maker; 1:Precipitation component of pET30a(+) induction;2:Precipitation component of pET30a(+) not induction;3:Precipitant component of pET30a(+)-Nek2 induction; 4:Precipitant component of pET30a (+)-Nek2 not induction;5:Supernatant component of pET30a(+) induction; 6:Supernatant component of pET30a(+) not induction; 7:Supernatant component of pET30a(+)-Nek2 induction;8:Supernatant component of pET30a(+)-Nek2 not induction (b) Expression of recombinant human Nek2 protein at different temperatures M:Marker; 1:After pET30a(+) induction; 2: pET30a(+) was not induced; 3:pET30a(+)-Nek2 was not induced; 4-7: pET30a(+) -Nek2 was induced at 18℃, 28℃, 37℃and 42℃ (c) Expression of recombinant human Nek2 protein at different IPTG concentrations M:Marker; 1:After pET30a(+) induction; 2:pET30a(+) was not induced ;3:pET30a(+) - Nek2 was not induced; 4-8: pET30a (+)-Nek2 was induced with 0.2mmol / L, 0.4mmol / L, 0.6mmol / L, 0.8mmol / L, 1.0mmol / L IPTG (d) Expression of recombinant human Nek2 protein at different times M:Marker; 1:After pET30a(+) induction; 2: pET30a(+) was not induced; 3: pET30a(+)-Nek2 was not induced; 4-12: pET30a(+) -Nek2 was induced at 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h 图2 重组人Nek2蛋白表达及条件优化 |
Fig.3 Gray value of the strip under different induction conditionsDifferent letters indicate P<0.05, the same letter indicates P>0.05 (a) Different temperature band gray value, F=1 339.962, P<0.05 (b) Different IPTG concentration band gray value, F=109.212, P<0.05 (c) Different induction time band gray value, F=138.483, P<0.05 图3 不同条件下电泳条带的灰度值 |
Fig.4 Recombinant human Nek2 protein purificationM:Protein marker; 1:Recombinant human Nek2 protein after purification 图4 重组人Nek2蛋白纯化 |
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Fig.1 The construction of pET30a(+)-Nek2 vectorFig.2 Recombinant human Nek2 protein expression and optimization of induction conditionsFig.3 Gray value of the strip under different induction conditionsFig.4 Recombinant human Nek2 protein purificationFig.5 Mass spectrometry identification of Mascot alignmentFig.6 Polyclonal antibody titer test resultsFig.7 Nek2 polyclonal antibody specific detectionFig.8 Nek2 subcellular localization/
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