A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C

BMC Biotechnology

Rajesh Kumar³  Raiees Andrabi³  Ashutosh Tiwari⁴  Somi Sankaran Prakash³  Naveet Wig¹  Durgashree Dutta³  Anurag Sankhyan³  Lubina Khan³  Subrata Sinha²  Kalpana Luthra³ 

[1] Department of Medicine, All India Institute of Medical Sciences, New Delhi, India

[2] National Brain Research Centre, Manesar, India

[3] Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India

[4] Present address: Centre for Biodesign, Translational Health Science and Technology Institute, Gurgaon, India

关键词: scFv;    V3;    Clade C;    HIV-1;   

Others  :  1134526
DOI  :  10.1186/1472-6750-12-87

 received in 2012-05-02, accepted in 2012-10-17,  发布年份 2012

【 摘 要 】

Background

Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.

Results

An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.

Conclusions

This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.

【 授权许可】

   
2012 Kumar et al.; licensee BioMed Central Ltd.

【 图 表 】

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