Biopanning of Anti c-Myc-tag Nanobodies and Its Application for Bioimaging

doi:10.13523/j.cb.20180209

China Biotechnology

2018, Vol. 38

Issue (2): 61-67 DOI: 10.13523/j.cb.20180209

Orginal Article

Biopanning of Anti c-Myc-tag Nanobodies and Its Application for Bioimaging

Jin-jing LI^1,²,Fei XU^1,²,Yan-wei JI^2,³,Mei SHU^2,³,Zhui TU³(

),Jin-heng FU²(

)

1 Institute of Life Sciences, Nanchang University, Nanchang 330031, China
2 Sino-Germany Joint Research Institute,Nanchang University,Nanchang 330047, China
3 State Key Laboratory of Food Science and Technology, Nanchang University,Nanchang 330047, China

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Abstract

Objective: Using c-Myc-GST protein as target molecule, nanobodies that can specifically recognize c-Myc tags (EQKLISEEDL) were screened from phage displayed immune libraries.Methods: Screening of phages specific bingding c-Myc-tag by solid-phase biopanning technology. Positive clones were identified by phage ELISA and then sequenced. The DNA fragment that coded positive nanobody phages were subclone to pET25b(+)vector ,the recombinant expression vector transformed into E.coli Rosetta (DE3) cells for expression under induction of IPTG. SDS-PAGE was used to analysis of recombinant protein expression. Finally, the binding activity and specificity of the nanobodies were confirmed by indirect ELISA and quantum dot immunofluorescence technology. Result: After four cycles solid-phase biopanning, phage with binding activity clones were effectively enriched, the recovery rate was improved by 145 times and positive rate increased from 20.83% to 85.4%. The nanobodies A25 and A26 with high OD₄₅₀ in phage-ELISA were recombinantly expressed , the production yield was 60 mg/L. Indirect ELISA results indicate that the recombinant proteins A25 and A26 can recognize the c-Myc-tag. Quantum dot immunofluorescence technology results showed that A25 could detect c-Myc protein in SP2/0 cells.Conclusion: Anti-c-Myc tagged nanobodies were successfully screened and two prokaryotic expression vector were constructed, the recombinant proteins achieved souble expression. And these laid the foundation for the detection of intracellular c-Myc protein.

Key words： Nanobody c-Myc Phage display ELISA Quantum dot

Received: 02 August 2017 Published: 21 March 2018

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	Yan-wei JI
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	Jin-heng FU

Cite this article:

Jin-jing LI,Fei XU,Yan-wei JI,Mei SHU,Zhui TU,Jin-heng FU. Biopanning of Anti c-Myc-tag Nanobodies and Its Application for Bioimaging. China Biotechnology, 2018, 38(2): 61-67.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180209 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I2/61

Table 1 Enrichment of phages in biopanning

Fig.1 The results of the specific positive clones identified by phage-ELISA

Fig.2 Indirect phage-ELISA for positive phage clones
PC: Positive control; NC: Negative control; BK: Blank

Fig.3 PCR products for recombinant plasmid colony of A25、A26
M: DNA Maker; 1 ~ 4: A25 recombinant colonies; 5 ~ 8: A26 recombinant colonies

Fig.4 Comparison of the deduced amino acid sequences of nanobodies

Fig.5 SDS-PAGE analysis of purification of nanobody
M: Protein maker; 1: A25 nickel column flowthrough; 2: A26 nickel column flowthrough; 3~4: Nickel column purified protein

Fig.6 Indirect ELISA for the protein
NC: Negative control; BK: Blank

Fig.7 QDs-SA-labeled mouse SP2/0 imaging


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