Related Experiment Video
Updated: Apr 7, 2026
Generation of Monoclonal Antibodies Against Natural Products
Published on: April 6, 2019
[Prokaryotic expression and polyclonal antibody preparation of antitoxin higA of Mycobacterium tuberculosis]
Dan Liu1, Zhengjun Yi2, Yurong Fu3
1Department of Clinical Laboratory Diagnostics, Weifang Medical University, Weifang 261053, China.
Objective:
To clone higA gene from genome of Mycobacterium tuberculosis, express higA protein in E.coli and prepare polyclonal antibodies against higA protein of Mycobacterium tuberculosis.
Methods:
HigA gene was obtained by PCR from the genome of Mycobacterium tuberculosis H37Rv strain and inserted into E.coli plasmid pET-32a⁺. The recombinant expression vector pET-32a⁺-higA was then transformed into prokaryotic expression bacterium E.coli BL21, and the recombinant protein higA was expressed in E.coli BL21 under IPTG induction. The protein was identified by SDS-PAGE and Western blotting. Purified protein higA was used to immunize New Zealand rabbits to prepare anti-higA polyclonal antibody. Quality and quantity of the antibody were examined.
Results:
HigA gene was successfully amplified and inserted into pET-32a.⁺ DNA sequencing confirmed the positive recombinant plasmid pET-32a⁺-higA screened by PCR and restriction enzyme digestion analysis. After induction with IPTG, the recombinant protein higA with 36.63 kDa was successfully expressed in E.coli BL21. Rabbit anti-higA polyclonal antibody with a good specificity was obtained, and the titer was above 1:3200.
Conclusion:
The higA protein was successfully expressed in E.coli BL21. Rabbit anti-higA antibody with high titer and specificity was prepared.
