Cloning Of Anti-CD45 Monoclonal Antibody And Analysis Of Their Sequences

Objective To clone anti-CD45 monoclonal antibody variable region gene fragments and analysis their sequences, lay the further foundation for modification of gene engineering antibodies study.Methods1. Total RNA extraction from hybridoma cell: use guanidine isothiocyanate-phenol-chloroform to extract the total RNA.2. Primer design: cloning human and mouse IgV gene by PCR method, selected the3 'end of primer complement with and CH1 areas, and the 5' end of primer and DNA encoding the signal peptide sequence complementary.3. McAbV gene amplification: RNA as a template, use cDNA synthesis kit for the first synthesis of the cDNA .And use cDNA as a template, with TaqDNA enzyme to amplificate McAbV gene.4. V gene nucleotide sequence analysis: Use gel extraction kit to purificate the PCR products, link McAbVH gene and VL gene with the pGEM-T vector, transfect into E. coli, and identify the bacteria by PCR-positive bacteria. Each connection product picks three positive bacteria to analysis the sequences.5. Use DNAtools, IMGT / QUEST and EBI TOOLS: ClustalW2 analysis software to compare the homology of the light chain and heavy chain gene respectively.Results1. The first time PCRUsing upstream primer (MH1, MH2) and downstream primer (IgG1-C 3') of two heavy chains; forward primer (MK) and reverse primer (Kc) of light chain for amplification, we obtained nucleotide sequence fragment of variable region that encoded monoclonal antibody. The length of variable region heavy chain (VH) and length of variable region light chain (VL) fragment including primer sequence was 397bp and 377bp respectively. In light of the amplified sequence fragment do not contain sequence of signal peptide and is suitable for construction of ScFV, further we changed another primer to amplify gene fragment including signal peptide sequence.2. The second time PCRUsing upstream primer (VH15',2,3) and downstream primer (IgG1-C 3') of three heavy chains; forward primer (VL5'1,2,3) and reverse primer(Kc) of light chain for amplification, we obtained 348bp functional nucleotide sequence fragment of variable region(VH), 57bp signal peptide sequence and non-functional fragment 369bp Vκ. To amplify functional light chain gene fragment we need re-design upstream primer that previous identified.3. The third time PCRUsing new synthesized primer VL5'4, 5 (instead of VL1), new synthesized downstream primer (VL3'1) for amplification, we obtained 333bp nucleotide sequence fragment and 57bp signal peptide sequence fragment. The sequence present functional after analysis with IMGT/QUEST.Classify the amplification of Ig gene , the result of anti-CD45 monoclonal antibody light chain V belong to the IGκV1-117'01 family and heavy chain belonging to the IGHV2-9-1'01 family. The amplification of a non-functional light chain belonging to the IGκV3-12'01 family.Conclusion We successfully obtained light chain and heavy chain gene sequence fragment and signal peptide gene fragment of anti-CD45 monoclonal antibody, laid the further foundation for modification of gene engineering antibodies study.