Construction Of Fully Human Lgκ Light Chain Loci By Large Fragment Operation Of Yeast Artificial Chromosome

Monoclonal antibodies play an important role in the antibody therapy of human disease and show an brillant prospect. However, because of their immunogenienty in human,the therapeutic use of hitherto developed monoclonal antibodies with mouse sequences, including chimeric antibodies, humanized reshaped antibodies and sigle chain FV fragment (miniantibodies) , were very limited.Introduction of unrearranged human germline immunoglubulin loci into mice in which the endogenous Ig genes have been inactived could be used to generate full human antibodies that show no immunogenicity in human and are of high affinity and specificity, This kind of research is of great significance for the elucidation of the mechanisms underlying programmed expression and assemblage of the human antibody. Meiotic homologous recombination between overlapping yeast artificial chromosome (YAC) can be used to reconstruct larger genomic segment in vivo. The present study is aimed at the construction of the gene of fully IgK light chain and the development of technical for the opration of large genomic segments.YAC host cell strain was first transformed with plasmid pRNA4 containing homologous sequences with the right arm of YAC and YAC with nutritional gene ADE2 in its right arm was obtained. The prepared YAC then be recombinated with another YAC containing longer homologous sequences using yeast meiotic homologous recombination method and a recombinant YAC of 190 kb containing invariable exons and enhancers, five J sections and four V sections was constructed. The two YAC used for experiment were from yeast CEPH and Olson pools.After the identification of the specific genes of recombinant YAC by Southern blotting, the obtained recombinant YAC was treated with Sail and Xbal and analyzed by Southern blotting with right and left arms of YAC, L13 and Ck as probes. The results showed that the prepared recombinant YAC contained repeated right or left arm fragments and the sequences of the genes involved in J and V sections became incomplete. These phenomena indicate that the results are not correspondent to our expectation. Despite of these, the present investigation was able to determine the positions and the directions of the genes in the recombinant YAC.The present study improved the protocols for yeast meiotic recombination and established basic techniques for the construction of fully human IgK light chain loci by large fragment operation of yeast artificial chromosome.