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Preliminary Study of Therapeutic Antibody Against Bacillus Anthracis

Author: LiBing
Tutor: ChenZuo
School: PLA Military Academy of Medical Sciences
Course: Microbiology
Keywords: anthrax chimeric antibody mammalian cell gene expression
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 70
Quote: 0
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Abstract


Anthrax is an infectious disease caused by spores of the bacterium, Bacillus anthracis. The only known effective pre-exposure prevention against anthrax is the anthrax vaccine. It takes a few weeks for individuals to produce neutralizing antibody, so it can not provide passive protection in the course of epidemic and emergency. Compared to the anthrax vaccine, the neutralizing antibody against protective antigen has advantage on providing direct and effective protection.The aim of the study is to express human-mouse chimeric antibody against anthrax protective antigen which can be used for a passive drug for the anthrax.Vl and Vh genes of the hybridoma cells lines 5E1, 4B2, and 2A8 were cloned by RT-PCR and further sequenced. All the Vl and Vh have the specific characterizations of mouse Ig after bioinformation software analysis.To further verify that the Vl and Vh have the biological activity, scFv-His were expressed in the E.coli cytoplasm. After purification by Ni-ATA, denatuation and renaturation by simply dilution, the purified scFv-His showed expected antigen binding activity and neutralizing activity. The above result lays foundation for expressing chimeric antibody.Full length H and L chain genes of chimeric antibody were constructed by fusing Vl and Vh with Cγ1 and Ck, respectively. The chimeric antibody expression plasmids were constructed by inserting full length H and L genes into the MCSs of the expression vectors. The plasmids constructed were transfected into the CHO/dhfr-(DG44) suspension cells. A few high yielding cell clones were obtained by enhancing stepwisely MTX concentration. A stable and high productive cell line with yield of 18 mg/L was obtained at MTX concentration of 5×10-8mol/L. The chimeric antibody was purified by ProteinA affinity chromatography and analyzed by non-reduced and reduced SDS-PAGE respectively. The molecular weights of the bands were consistant with the expected that is 150kD on the non-reduced gel and 50kD and 25kD on the reduced gel. Western blot showed that both the intact antibody molecular and H chain can specifically react with anti-human IgG Fc. ELISA showed that the chimeric antibody has similar capacity of binding with rPA antigen compared to the mouse mAb. The purified chimeric antibody also showed strong neutralizing activity on both the cell model and animal model. Chimeric antibodies may be provided for potential application as a passive immunization therapeutic agent in cases of B. anthracis exposure and infection.

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