Using Phage Antibody Library Screening Her2 Antibody

Malignancy is the most active field in the studying and exploring of therapy antibodies. Recently people have payed more attention to the therapy antibody for its advantages such as particularly effects, few harmful side effects.The gene of the receptor ligand domain 2 of HER 2/neu (RLD 2) was amplified by PCR, cloned into the pET-24a(+) expression vector and expressed in E.coli. After purified, the concentration of the protein was 1.05 mg/ml and the purity was over 95 %. Screen the antibodies of anti-HER 2 from the natural phage library by antigen-coated phage selection. Four rounds of selection against RLD 2 showed specific enrichment of phage antibody. After the fourth round of selection 28 out of 46 clones exhibited binding capacity. Select 6 clones which had binding activity to further identify the specificity. The result showed that 6 clones had high binding capacity and specificity to RLD 2 while had no binding activity to BSA, HPA-8 kD, mHPO, EGF and LCC.Human breast tumor MCF-7 which had low expression of HER 2, human breast tumor SKBR-3 cells which had high expression of HER 2, Balb/c-3T3 and ECV 304 were used to determine the activity of antibodies in vitro. The result showed that 6 antibodies could inhibit the growth of SKBR-3, but they had no effect on MCF-7 and ECV 304. We select two clones, and found that they could inhibit the growth of Balb/c-3T3.We hoped to express the Fab of the antibodies in E.coli. Digestion of the vector with Fab fragment with Nhe I and Nde I to remove of the GeneIII. The cohesive ends were blunted and ligated, and then the product was electrotransformated in E.coli. After removement of the GeneIII, we added 6 histidine following the Fd fragment in order to identify it and purify it. The experiment to improve the expression of the antibody is going on.