Construction Of Humanized Antibody For Mammalian Expression

Antibody is Y-shaped protein molecules capable of recognition and discrimination of hundredsof millions of self and non self substances in a host organism. Abs is the central component ofadaptive immune system and can be found on the membrane of B-cells, or in blood, in a solubleform. Humanized antibodies were successfully expressed through a diversity of mammalianexpression vectors that have been generated and used. The mammalian expression system is thebest host. The approach in the study of the antibodies has been one of the focal points of biologyand modern medicine. Several diseases are with a new hope since the treatment using antibodiesis being developed by genetic engineering. The capacity that the antibodies have in associating achemical structure predetermined, has stimulated the emergence of new drugs. The mainobjective of this study was to construct humanized antibody with a mammalian expression vectorpEGFP-C1into competent E. coli cells. The Oligonucleotide used in the reaction was designedfrom the sequence H1c9permitting an insert a cleavage site for restriction enzyme BglII at the5’end and EcoRI endonuclease site to the3’ end of the gene to facilitate cloning posteriors. Themammalian expression vector was pEGFP-C1. Polymerase chain reaction product and restrictiondigest were electrophoresed on1%agarose gels containing ethidium bromide. Humanized singlechain fragment variable fragments of the expected size were excised from the DNA was purifiedusing a gel extraction Kit (Nucleic acid purification columns). Plasmid DNA from ligationreactions was to transform chemically competent E. coli. In conclusion, with all the dataobtained from my research work, it is revealed that humanized pEGFP-C1-hscFv vector wasconstructed successfully.