Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein

doi:10.13523/j.cb.1908061

China Biotechnology

2020, Vol. 40

Issue (3): 31-37 DOI: 10.13523/j.cb.1908061

Orginal Article

Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein

CHEN Qiu-li¹,YANG Li-chao¹,LI Hui¹,WEN Sha¹,LI Gang¹,HE Min^1,^2,^**(

)

1 School of Public Health Guangxi Medical University, Nanning 530000,China
2 Guangxi Medical University Laboratory Animal Center,Nanning 530000,China

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Abstract

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.

Key words： Nek2 Prokaryotic expression Polyclonal antibody KCl stain

Received: 30 August 2019 Published: 18 April 2020

ZTFLH:

Q816

Corresponding Authors: Min HE E-mail: hemin@gxmu.edu.cn

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	Qiu-li CHEN
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CHEN Qiu-li,YANG Li-chao,LI Hui,WEN Sha,LI Gang,HE Min. Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of Human Nek2 Protein. China Biotechnology, 2020, 40(3): 31-37.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1908061 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/31

Fig.1 The construction of pET30a(+)-Nek2 vector M:DNA marker; 1:PCR amplification product of Nek2 gene; 2:Colony PCR detection; 3:Bam HI and Sal I enzyme digestion of recombinant plasmid

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

Fig.3 Gray value of the strip under different induction conditions Different 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

Fig.4 Recombinant human Nek2 protein purification M:Protein marker; 1:Recombinant human Nek2 protein after purification

Fig.5 Mass spectrometry identification of Mascot alignment

Fig.6 Polyclonal antibody titer test results

Fig.7 Nek2 polyclonal antibody specific detection

Fig.8 Nek2 subcellular localization


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