| Antibodies were widely used in the clinical medicine. Most sources of them are extracted from the human blood and mouse monoclonal antibodies. But even with the most vigilant of preventive measures, an arrays of the potentially deadly infections agents such AIDS or hepatitis continuous threaten to contaminate the plasma products of serum, and the mouse monoclonal antibodies application was limited as their immune reaction. To reduce these risks, the humanized monoclonal antibodies were carried out using combination of recombinant DNA technique, but they cannot be applied extensive due to their low yield and affinity, difficult to purification and expensive. At the beginning of 1990s, the mice in which the endogenous heavy and kappa light chain loci have been knock out and can generate repertoires of fully human antibodies was obtained. Now the cloned cattle expressed human antibody have been produced with the development of large-animal transfer technology, but the presence of active endogenous bovine Ig loci may interfere with the expression of the human antibodies, hence, knocking out or in some other way inactivating IgH or Igλ. loci was required first. Based on the experience of creation of mice perform a human repertoire and the characteristic of bovine Ig heavy chain, we construct a gene targeting vector to knock out the bovine JH functional exons and E μ gene.In this experiment, genomic DNA was extracted from Holstein bovine liver tissue, PCR primers were designed based on the genes, which have been deposited on NCBI GenBankTM. Three fragments: 1.12kb (J), 3.63kb (Ml), 1.12kb (M2) were amplified from bovine genomic DNA. The J fragment including the first, second and third exons (unfunctional) of JH locus; Ml fragment including the Cμ CH1, CH2, CH3, CH4; TM1 exons; M2 including partial sequence of Cμ-Cσ intron. Comparison these products sequences with the corresponding gene submitted on NCBI GenBank? showed that the homologous of them exceeding 99%. It is demonstrated that they are all conserved genes and adapt to use as homologous arm in the targeting vectors.Using J as short arm, Ml, M2 as long arm, the targeting vector pGTN29-TK-J-M 1-M2 was constructed. The positive selection marker neo was placed in the middle of short and long arm and the negative selection marker tk was just outside the long arm.The linearized targeting vector DNAs were introduced to the bovine fetal fibroblast cells by Lipofectamine or alcium phosphate methods, 123 clones were picked up after cultured in the G418 and Gancyclovir for two weeks. The clones were amplified and identified by PCR and six positive clones, which may take place correct homologous recombination evens, were obtained.The limited germline sequence diversity both at the heavy and light chain loci, especially only one VH family was used preferential imposes constrains on generation of combinatorial diversity in cattle, the cattle thus must employ other strategies during evolution for antibody diversification.Using PCR, RT-PCR and Southern analysis, we found the two JH genes (accession no. AY 158087, AY 149283) and two C μ genes (accession no.AY230207, U63637) all exit as two copies genes in the bovine germline. Although they are functional genes, the expression levels between them are distinctlydifferent. A BLAST search of the NCBI GenBankTM and bovine EST data bases suggested that the JH gene (accession no. AY 158087) and C μ gene (accession no. AY230207) are the only used genes in the reported cDNA sequences. The PCR results also indicated that the JH gene (accession no. AY 158087) is in series with C μ gene (accession no. AY230207) and the JH gene (accession no. AY 149283) is in series with C μ gene (accession no.U63637). Interestingly, there is a shortened lack of 1.5kb S μ sequence occurred in the last combination. This may be the reason that it expressed at low level. Two copies of JH and C μ genes in the bovine germline may contribute to the antibody diversification as well.
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