Cloning And Sequence Analysis Of The Bovine Immunoglobulin Lambda Light Chain Constant Region

Immunoglobulin is very important for human body to resist the pathogens such as bacteria and virus. Human polyclonal antibodies (hPABs) are useful therapeutics to treat many diseases, but they are available only from human donors and their supply and application is limited. Transgenic cattle carrying hIg loci could be useful for large volume commercial production of hPABs, which can be used in clinic therapia especiallary for the difficult diseases such as AIDS and cancers. Transgenosis comprises knocking -out bovine Ig genes in livestock and knocking- in hIg gene loci.The focus of the dissertation work is to gain some sequence information about the bovine immunoglobulin lambda light chain constant region. Several positive clones containing the bovine C_λ gene segment were isolated from bovine genomic DNA library. About 15kb germline DNA sequence was sequenced. The research work including:1. A bovine genomic λ-phage DNA library and a bovine genomic bacterial artificial chromosome (BAC) library were screened with a probe specific for immunoglobulin lambda light chain loci. Two recombinant phage clones containing the C_λ gene segment were isolated from the λ-phage library and the insert segments were 7kb and 9kb respectively. One positive BAC clone was isolated and pulse field gel electrophoresis (PFGE) result verified that the insert size was about 130kb.2. The BAC clone DNA was digested with eight different restriction enzymes and subjected to Southern Blot analysis by hybridization with a 408bp C_λ probe. Seven positive lanes were presented at the most. The result indicated that there were at least seven C_λ segments in the bovine immunoglobulin lambda light chain constant region.3. Five positive lanes isolated from the clones were cloned into the pGEM-3Zf(+) vector and the recombinant plasmid were named pGEM-3zf2.8, pGEM-3zfSl, pGEM-3zfS2, pGEM-3zfS3 and pGEM-3zfS4. The insert segments contained the C_λ gene were sequenced and the length of them were 2660bp, 1682bp, 2514bp, 1860bp and 1806bp respectively.4. Constant region-specific PCR were conducted to investigate how the positive sequences we got arranged using several different primers. The PCR products indicated that the segments S3, S2 and S4 aligned in the lambda light constant region and span about 15kb distance.5. Comparing fifteen C_λ sequences, including four bovine, seven human and four mouse IgC_λ, we have mapped the evolution tree of the lambda light chain constant genes. The homology between the bovine and the mouse was higher than that between the bovine and the human. The bovine and the mouse maybe have closer relative relationship from evolvement aspect.