Production Of Monoclonal Antibodies Against CD55 Molecules By DNA Immunization Followed By A Single Boost With Cells And Study On Biological Activity Of Mab

Objective: To Produce Mab against human CD55 molecules by DNA immunization followed by a single boost with cells. And to determine practicability of this means to produce Mab base on the biological activity of CD55 antibody. Methods: The fμl 1-length gene of human CD55 molecules was amplified by reverse transcriptase polymerase chain reaction (RT-PCR).The fμl 1-length gene CD55 was cloned into the expression vector pcDNA3.1 and pET28a. CD55 was expressed in E.coli Rosseta by inclusion body. The inclusion body was denatured by guanidine hydrochloride and was purified by Ni+ column. The plasmid pcDNA3.1/55 was injected into mice quadriceps muscle of thigh and we pretreat muscles as followed: (1) inject cardiotoxin on day 7 before DNA immunization. (2) inject high osmotic pressure sucrose solution. For boost the CD55 immunized mice, HPB-ALL high expressed CD55 molecules cell lines were injected on day 3 before fusion. Serum was drawn on days 10 after DNA immunization every time for evaluation of the anti-serum titer.Then monoclonal antibody was prepared by conventional hybridoma technique. The isotype of antibody was detected by sandwich ELISA. And simple topographical epitope was mapped by immune compete. To identify the affinity of antibody to nature membrane protein and denatured protein, we utilize FASC, fluorescence microscope and Western blot respectively.Result: (1) The 1174bp fμl 1-length CD55 gene was amplified, and the recombinant plasmid pcDNA3.1/55 was constructed by cloning the 1174bp gene into pcDNA3.1. (2) The highest anti-serum titer was 1:200 after DNA immunization three times, and...