The Screening And Expression Of Human Antibody Against Human β Amyloid Peptide In Alzheimer's Disease

Alzheimer's disease (AD) or senile dementia is an age-related neuro-degenerativedisease. At the end of last century, the population of our country has entered into an agingsociety in advance. Alzheimer's disease is becoming the fourth mortality cause of human, being secondary to heart disease, tumor and brain stroke. Up to date, the diagnosis andtherapy of Alzheimer's disease remain a problem.We used the synthesized amyloid P peptide 40 (A P 40), which is involved in the pathogenesis of Alzheimer's disease, and its amino-end 16 amino acids fragment (A β 16) as antigens to screen a human artificial synthesized single-chain antibody library separat(?)ly. After five rounds of amplification, absorption and elution, 53 and 55 clones were randomly picked from the agar plates. ELISA testing identified 8 and 33 candidate clones with binding activities to A P 40 and A β 16 respectively. Among them, only 10 clones were inhibited by 100 nanogram of A β 40 to below half level of its original binding activities, which meant they were specific phage antibody clones of A P 40. In addition, 5 clones of them passed the competitive ELISA testing to A β 16. So, the phage antibodies from the five clones recognize the epitope between amino-end 16 amino acids of A β 40.We designed a pair of specific primers to amplify the single-chain fragment variable (scFv) gene from the four positive clones by polymerase chain reaction and cloned into the pGEM T easy vector. DNA sequences of scFv specific to A P 40 were consisting of 768bp determined by ABI 3700 sequencer with T7pro and SP6 primer. The deduced amino acids sequence confirmed its typical human antibody variable region characteristics, then the complement determinant regions (CDRs) and framework regions (FRs) were discriminated empirically. The scFv gene was registered in GenBank with No.AF502169. Blast analysis demonstrated that the gene of heavy chain variable region was 97% homologous to contig clone 14-NT010168.11 and the gene of light chain variable region was identical to 22-NTOl 1520.8. These mean that the genes of human antibody heavy chain and light chain specifically to A P 40 are deriving from human chromosome 14 and 22 respectively, which is identical to the known data of human antibody gene. The light chain of human antibody against A P 40 is λ- type. Compared with other single-chain antibodies from the same library, the amino acids sequence diversity are mainly located within heavy chain variable region, which imply the importance of amino acids sequence of antibody heavy chain in...