Development Of Recombinant Human Iga Momoclonal Antibody Against Alpha-Crystallin Of Mycobacterium Tuberculosis

Mycobacterium tuberculosis (MTB) is one of the world’s most hazardous pathogens. About thirty percent people are infected by MTB in the world, among which ninety percent people become latently infected. Alpha-Crystallin (Acr) is expressed by hspX (Rv2031c,acr) gene of mycobacterium tuberculosis. Many reports demonstrated that this protein was closely related to MTB dormancy. Some studies have confirmed that alpha-crystallin was a target antigen against TB. In this study, we will obtain the specific human scFv against mycobacterium tuberculosis (MTB) alpha-crystallin (Acr) protein using phage-displayed antibody library technology. Full length heavy and light chain genes of IgA were constructed for production of intact IgA antibodies in Chinese hamster cell lines. This work paved the ground for the study of IgA antibodies for the control of mycobacteria tuberculosis.To express the MTB Acr protein in prokaryotic system, the acr gene was inserted to expression vector pCold to construct the recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21(DE3) and was induced with IPTG. SDS-PAGE and Western blotting analysis showed that the expressed recombined protein had a molecular weight of19kDa. The Acr protein was expressed as soluble protein under the condition of low temperature. The concentration and the purity of the protein are0.8mg/ml and90%respectively after purification.A human large phage antibody library was panned four times with the purified Acr protein. Soluble scFvs were prepared through infection of E.coli HB2151with the selected phage antibodies clones and induction with IPTG. The antigen binding activity were determined ELISA and Western blotting, and DNA sequences of these clones were analyzed by Sanger sequencing.43positive clones were obtained after4rounds of panning and29clones had binding ability to MTB Acr protein. DNA sequencing of the29clones showed26different positive clones. After infection in E.coli HB2151we obtained14specific soluble scFvs. DNA sequence analysis showed that the variable regions of these scFvs belonged to different subgroups. The Western blotting results suggested that the anti-Acr antibodies had good immuno-reactivity with natural Acr protein.We constructed full length heavy and light chain genes of anti-Acr IgA by fusing heavy chain variable region (VH) and light chain variable region (VL) with heavy chain constant region of IgA2and kappa chain constant region, respectively. The antibody expression plasmids were constructed by inserting full length heavy and light genes into the multiple cloning sites of plasmid pEF-dhfr-Neo. The full-length light and heavy chain expressing plasmids pEF-dhfr-IGH and pEF-dhfr-IGK were co-transfected into the CHO/dhfr-cells,72hours later we harvested IgA in cultured cell supernatants and used IgA(α)、kappa mAb and MTB Acr protein to detect its expression. Cell lines expressing anti-Acr IgA were constructed.In this study we expressed MTB Acr protein in soluble form in E.coli, and by panning the human large phage antibody library, we obtained14specific antibody clones. The Western blotting results suggested that the anti-Acr antibodies had good immuno-reactivity with natural Acr protein. We constructed IgA light and heavy chain eukaryotic expressing plasmids and expressed full-length anti Acr IgA in Chinese hamster cell lines. Subsequent study suggested the recombinant full-length anti-Acr IgA antibody exhibited specific MTB Acr binding activity.