Anti-a¦Â Human Single Chain Antibody Gene Screening And Antibody Gene Expression

Alzheimer's disease(AD) is a degenerative disease of the central nervous system, progressive dysmnesia and intelligent recession as main clinical feature. It is neuropathologically characterized by the deposition of extracellularβ-amyloid (Aβ)-containing plaques, intracellular neurofibrillary tangles, reduced synaptic density and neuronal loss in selected brain areas. The aggregation of beta-amyloid (Aβ) peptides in the senile plaques is believed to contribute to neuronal dysfunction and death, and impairment of memory in AD patients. Research on preventing Aβassembly, reducing parenchymal plaque burden, decreasing total Aβbrain levels and reversing cognitive deficits has become valuable. Anti-Aβantibody studying provide a hopeful approach for AD therapy. In animal trials, direct administration of anti-Aβantibodies has proved successful in clearing amyloid from the brain and reversing memory deficits. But the report of successful clinical use of anti-Aβhumanized antibodies to stimulate Aβclearance hasn't been found.Our study is dedicated to preparing humanized scFv antibody agaist Aβ_1-42 by phage display technic and constructing entire antibody gene. Two parts is included: first, to screen out the specific antibody clones againstβamyloid peptide 1-42 from human phage-display single-chain Fv antibody library, clone the antibody gene and express it in a bacterial system, and confirm its antigen-binding ability and biological activity. Second, to construct IgG entire antibody gene and entire scFv gene, and express them in a eukaryotic system.1. Screening, bacterial expression and characterization of human scFv antibody against Aβ1-42 peptide.βamyloid peptide 1-42 was bound on the solid surface of 96 wells plate as antigen to screen the binding clones from a human phage-display scFv antibody library. After four rounds of "binding-elution- amplification", a large enrichment was observed. 80 picked randomly well-separated colonies were identified by ELISA test. The results showed that 7 positive colonies had remarkable binding abilities with highest OD490 value and none of cross-reactivity with other kinds of antigen. The specific positive phage clones were infected into the host E. coli HB2151 to express soluble scFv antibodies and three different kinds of extract (culture medium, periplasmic space and whole cell extract) were prepared after IPTG induction. The soluble scFv antibody from clone A10 was expressed successfully and identified by SDS-PAGE and western blot, and antigen-binding activities were determined by ELISA. The results of SDS-PAGE and western blot showed a new protein band with a proximate MW of 33KD was mainly in whole cell extract. The value of the expression products was five folds as that of control by OD 490nm test, and none of cross-reactivity with BSA. The results of biological activity showed the scFv antibody could bind to Aβfibrella in senior plaques of AD patients' brains. The positive scFv antibodies gene was sequenced. DNA sequencing data demonstrated that the gene of the positive antibody specifically against Aβ1-42 was the scFv gene and the deduced amino acids sequence confirmed its typical antibody V domain structure. 2. Construction and eukaryotic expression of entire antibody gene.At first, we synthesized H and L signal peptide gene obtained from NCBI blast results. Then we amplified the target antibody gene by using specific primers. We acquired IgG entire antibody H gene(H signal peptide gene +VH gene +CH gene), IgG entire antibody L gene (L signal peptide gene +VL gene +CL gene) and entire scFv gene (H signal peptide gene +scFv gene +FC gene) by performing overlap PCR, then cloned the gene into pcDNA3.1 vector respectively. The results of PCR and DNA sequencing data showed three kinds of constructed gene confirmed their expectant DNA sequence. IgG entire antibody H gene-vector plasmid and IgG entire antibody L gene-vector plasmid were transferred to CHO cell together with the purpose to produce IgG entire antibody. Entire scFv gene-vector plasmid was transferred to CHO cell so as to produce scFv entire antibody. Cell culture medium ELISA test results showed negative. Condition optimation may be needed. In the study, we have set a basis for further study, including expression optimation, animal test and exploratory development.So far, some reports on screening and characterization of human scFv antibody against Aβpeptide have been found, but they are in phase of lab research. The report of successful clinical use of anti-Aβhumanized antibodies and entire antibody preparation hasn't been found. NCBI blast shows our obtained anti-Aβhumanized antibody gene sequence is a new sequence. Our study will be a powerful tool for AD immunotherapy and clinical use study.