| The shortcome in clinic treatment of mouse monoclone antibody is the immunogenicity in human.The method to solve these problem is to humanize mouse monocloal antibodies.The course of humanize mouse monocloal antibodies has four steps: human and mouse chimeric antibody ,the humanized antibody ,the antibody from phage antibody library,the human antibody expressing from transgenic animnals.The antibody expressing from transgenic animnals has advandage:In one hand ,it hasn't immunogenicity;On the other hand ,The transgenic animnals can expressing antibody of human antigen, this will help to cure the disease of self-immunity.The advance of humanized antibody is transgenic animnals expressing human antibody Five-features translocus mice which established by British Brabaham Institute expressing human Ig is different from those constructed by Abgenix company in USA and Kirin company in Janpan .Except for mice Ig heavy chain and k light chian knocked out, knock-in human Ig heavy chain, K light chian, human Ig A. light chian had also knocked in.But human Ig heavy chain in Five-features translocus mice was too short ,it's 3 constant chain only contain C μ and C δ without C γ .So Five-features translocus mice expresses human IgM only without IgG, Comparing with IgM , IgG has high expression in amount, It also has effective function and higher affinity.So it is important for the Five-features translocus mice to be reconstructed to expressing human IgG after stimulating by antigen,In order to obtain the proper gene fragment, The phage human genomic DNA library have been screened,Because phage vector contains 9-20kb forgein gene fragmentjust as the length as needed.So during screening the phage human genomic DNA library, the conserved methods was employed:First, the human genomic DNA phage library has been amplified the signal for screen easyly.The Amplified sub- library was identified by both primers of human code sequence of IgG2 and IgG3 to ensure the positive sub-library contains both the IgG2 and IgG3 gene fragment in the phage . Second:In order to obtain the whole sequence needed ,the experiments screen the positive sub-library with both primer pairs of IgG3 located at promotor and the code sequence to ensure obtaining the full length monoclone.Last :During the screening of the library ,the Isotop P- was used only once,and then the full length monoclone was obtained after many times PCR.The full length monoclone phage vector contains 11.2kb C γ 2 gene which included:promoter(P) the first translate exon (I) switch area (S) coding sequence(C) two trans-member exon(MK M2) and its PolyA signal (PA) ,these are the integrate for class switching .The sequence of Cγ2 screened has not yet published on genebank.The constructed yeast targeting vector pcDNA3-γ2 consists of Cγ2 mice-origintedenhancer, selecting gene G418 ,left and right homologous sequence . The gene-targeting vector pcDNA3- Y 2 validated by assays of restriction enzyme digestion, sequencing,The gene-targeting vector pcDNA3-γ2 was used to target the YAC which contain six V genes all the D and J genes μ chain δ chain of the human heavy chain .The result of the experiment shows that:The lithium acetete transformation is more effect than electroporation. The reason of negative result are dicussed.
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