Characterization of heavy-chain antibody gene repertoires in Bactrian camels

Camelids are the only mammals that can produce functional heavy-chain antibodies(HCAbs).Although HCAbs were discovered over 30 years ago,the antibody gene repertoire of Bactrian camels remains largely underexplored.To characterize the diversity of variable genes of HCAbs(VHHs),germline and rearranged VHH repertoires are constructed.Phylogenetics analysis shows that all camelid VHH genes are derived from a common ancestor and the nucleotide diversity of VHHs is similar across all camelid species.While species-specific hallmark sites are identified,the non-canonical cysteines specific to VHHs are distinct in Bactrian camels and dromedaries compared with alpacas.Though low divergence at the germline repertoire between wild and domestic Bactrian camels,higher expression of VHHs is observed in some wild Bactrian camels than that of domestic ones.This study not only adds our understanding of VHH repertoire diversity across camelids,but also provides useful resources for HCAb engineering.

Expression of heavy-chain variable domain of mAb against Encephalitis Type B virus in transgenic tobacco

SINCE the first report on immunoglobulin gene expression in plant by Hiatt et al. in 1989,analogous researches have been further developed. Up to now, it has become a new method touse transgenic plants and plant cells to produce antibodies and study disease-resistance breedingof crops, plant metabolism and growth, which is an organic combination of immunologyand plant sciences at molecular level. According to recent reports, both higher and lowerplants, such as tobacco, Arabidopsis, calli of maize and unicellular green alga, can

Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

In this study,we introduced the bovine immunoglobulinμheavy-chain gene(the orphaned gene on BTA11)into mouse germline cells.Bovine IgM was highly expressed in selected transgenic lines,and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain(IgH)genes in these lines.The forced expression of bovine IgM resulted in reduced numbers of pro-and pre-B cells but increased the number of immature B cells in the transgenic mice.Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen,but most of the cells are arrested at the T1 transitional B cell stage,leading to a significantly lower number of T2 transitional and mature B cells in the spleen.Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased,the IgA levels were slightly increased compared to the WT mice.The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM,suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells.These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3(CDR3)sequences in their VH repertoire and Vκrepertoire but exhibited an increased frequency of unique CDR3 in their Vλrepertoire.Compared to the WT mice,the transgenic mice had a significantly higher percentage of mouse IgMexpressing B cells that expressedλchains.Finally,we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.

Isolation and optimization of camelid single-domain antibodies:Dirk Saerens'work on nanobodies

It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain homodimer.These so-called heavy-chain antibodies(HCAbs)are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol.These HCAbs are easily purified from serum,and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies.The antigen binding site of the dromedary HCAb comprises one single domain,referred to as VHH or nanobody(Nb),therefore,a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens.The monoclonal Nb is produced well in bacteria,is very stable and highly soluble,and it binds the antigen with high affinity and specificity.Currently,the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors,to diagnose infections,or to treat diseases such as cancer or trypanosomiasis.

A comprehensive analysis of immunoglobulin heavy chain genes in the Bactrian camel(Camelus bactrianus)

Heavy chain only antibodies(HCAbs)represent a rare type of antibody that is devoid of light chains and the CH1 domain that have been reported in cartilaginous fish and camelids.By analyzing transcript data and genome sequences,we conducted a comprehensive analysis of Bactrian camel immunoglobulin heavy chain genes.Based on the transcript data,oneμgene,fiveγgenes,oneαgene and oneεgene were found.Additionally,the variable region of HCAbs(VHH)and the conventional antibodies(VH)sequences associated with theγ3,γ1a/b andμgenes were amplified.Based on these genome sequences,seven DH,six JH,μ,γ2a,γ2c,α,andεgenes and a portion of aγ3 gene were observed.Different Kozak sequences within different VH families were found in our analysis,and the variability index differed between the VHH3 and VH3 families.Phylogenetic analysis of the constant regions of the camelid immunoglobulin genes indicates that these genes appeared before the evolutionary divergence of Bactrian camels and dromedaries.