Reconstruction Of MCS Of PTriEx-4 Neo Vector Sequencing Of Variable Region Gene Of Anti-TM-TNF-αMonoclonal Antibody C1 And Construction Of Chimeric Antibody

In modern scientific research of certain target gene, we often need to express the same gene within different expression vectors tailored to fulfill diverse experiment purposes and experiment systems. When large amouts of pure protein is needed, the gene of interest must be inserted into prokaryotic or insect expresson system with purification tag. When the function of gene in cells is under investigation, the gene of interest must be inserted into eukaryotic expression vector. However, MCS from different vectors differs from each other significantly, when expressed in different vectors, we have to conduct primer design, primer synthesis, PCR, digestion, ligation, transformation, screening and sequencing all over again, which is labor intensive and demanding; in addition, there may be mutations introduced into gene of interest during PCR, making all the procedures above futile.To solve this problem, we design strategy for construction of series of vectors. The basic idea is to supplant all Multiple Cloning Site of different vectors with the MCS of pEGFP- C1. Because the MCS of pEGFP- C1 is too short to recycle, so we need to insert a small fragment about 238bp into it at the EcoRⅠsite. After removing the 238bp fragment, the vector was successfully constructed. The reconstruction of MCS of pTriEx-4 neo is a part of reconstruction of expression vectors project: Recostructionof the MCS of the pTriEx-4 neo1. Construction and cloning of pTriEx-4 neo/MCS-238 recombinants:Using the plasmid pEGFP-C1/238 as template, the MCS of pEGFP-C1 with 238bp in it was amplified by PCR and EcoR V and Bsu36 I restriction sites was introducted at the same time. After digestion with EcoR V and Bsu36 I, the the fragment of 320bp was inserted into pTriEx-4 neo plasmid by T4 DNA ligase. Then the recombinant pTriEx-4 neo/MCS-238 was transformed into E. coli DH5αand the positive clones were screened by Ampicillin resistance.2. Identification of positive clones: The positive clones were confirmed by different way, the result showed that: the recombinants are digested by EcoR V and Bsu36 I digestion, showing the cleaved fragment from the recombinant with the molecule weights of 320bp, suggesting pTriEx-4 neo/MCS-238 plasmid was successfully constructed.3. Digestion of 238 bp with EcoRI and construction of pTriEx-4 neo/C1 recombinant:TriEx-4 neo/MCS-238 was digested by EcoR I, and then connected by T4 DNA ligase. The recombinant was transformed into E. coli DH5αand the positive clones were screened. The positive clones were confirmed firstly by colony PCR, one positive clones selected for sequencing. DNA sequence analysis showed that MCS of pTriEx-4 neo/ C1 have been successfully replaced by the MCS of pEGFP-C1 and no shift and mutation. Conclusion: This study had replaced its original MCS of pTriEx-4 neo with the MCS of pEGFP-C1 successfully. In order to standardize the MCS of pEGFP-C1,pET-28a(+), pcDNA3.1(+)and pTriEx-4, this study completed the work partly. The research of reconstruction of MCS provides an effective experiment tool and system for further study which based on the molecular cloning. Tumor necrosis factor alpha (TNF-α), a pleiotropic cytokine with a wide range of biological activities, plays important roles in regulation of immune response and inflammation and also governs cell differentiation, proliferation and apoptosis. There are two forms of TNF-α, namely secreted TNF-α(S-TNF-α) and transmembrance TNF-α(TM-TNF-α). TM-TNF-αis the precursor of S-TNF-α. TNF-alpha converting enzyme (TACE) cleaves TNF- alpha within the extracellular domain of TM-TNF-α, releasing soluble TNF-αfrom cells. TM-TNF-αand S-TNF-αhave different bioactivities. Excessive S-TNF-αproduction is one of the most significant manifestations under pathological conditions such as rheumatoid arthritis and Crohn's disease.Our lab successfully obtained a new anti-TM-TNF-αmonoclonal antibody, which only binds TM-TNF-α, while displays no affinity to S-TNF-α. Actually, this new mAb recognizes the TM-TNF-αcleavage site selectively, blocking TACE enzymatic action through spatial hindrance. The beauty of this mAb lies in its potential to prevent TACE from shedding S-TNF-αinto extracellular fluid without interfering with the functional domain of TM-TNF-α.One of the major impediments of murine mAb clinical application is its immunogenicity on human body, which makes chimerization and humanization necessary for modern therapeutic mAbs. This project is aimed at sequencing variable region gene of mAb C1 and construct chimeric antibody, paving the way for further humanization.1. Cloning and sequencing of light chain variable region gene of mAb C1 Employing family specific mouse immunoglobulin light chain leader sequence degenerate primer combined with framework region 4 degenerate primer, we have successfully amplified light chain variable region gene of mAb C1 by polymerase chain reaction. The C1-VL5'2 sequence is confirmed to be a new functional productive light chain variable region gene that has undergone in frame rearrangement of V/J segments without stop codon by online immunoglobulin V-region analysis and alignment bioinformatics tools from NCBI IgBLAST and IMGT V-quest.2. Cloning and sequencing of heavy chain variable region gene of mAb C1Employing family specific mouse immunoglobulin heavy chain leader sequence degenerate primer combined with framework region 4 degenerate primer, we have successfully amplified heavy chain variable region gene of mAb C1 by polymerase chain reaction. The C1-VH5'1 sequence is confirmed to be a new functional productive heavy chain variable region gene that has undergone in frame rearrangement of V/D/J segments without stop codon by online immunoglobulin V-region analysis and alignment bioinformatics tools from NCBI IgBLAST and IMGT V-quest.3. construction of C1 chimeric antibody expression vector and expression of C1 chimeric antibodyBy overlapping extension polymerase chain reaction, we fuse light chain variable region gene of mAb C1 with human kappa constant region gene, which is inserted into multiple cloning site A of pIRES vector; we also fuse heavy chain variable region gene of mAb C1 with human gamma 1 constant region gene, which is inserted into multiple cloning site B of pIRES vector. Detection of chimeric antibody in supernatant of pIRES-C1 expression vector transfected CHO cell culture by sandwich ELISA leads to positive result, indicating successful construction of C1 chimeric antibody expression vector and secretion of chimeric antibody by pIRES-C1 transfected CHO cells. Conclusion: We have successfully cloned light chain and heavy chain variable region gene of anti- TM-TNF-αmonoclonal antibody C1, which is confirmed to be a new pair of functional rearranged variable region gene by immunoglobulin sequence analysis. By overlapping extension polymerase chain reaction, we successfully construct pIRES single promoter bicistronic chimeric antibody expression vector and express chimeric antibody in CHO mammalian expression system.