Construction Of Anticalin Library And Screening Antiampicillin Mimic Antibody By Ribosome Display

Ampicillin is one ofβ-lactam antibioties. It is one of commonly used drugs that treat of dairy and other food animal diseases. Will ampicillin residues in the animal foods can cause the huge harm to the body, environment or the industrial production. World Health Organization reports that the incidence of side effects by using ampicillin is second in the all drugs. The analytical method of immunology is economic, simple and rapid, but the specific antibodies are difficult to obtain, because of difficult to synthesis antigen and long time of immune. The lipocalin scaffold has emerged as an adaptable receptor for small molecule binding. Anticalin is a novel class of engineered receptor proteins with prescribed ligand specificity derived from the lipocalin scaffold. Anticalin may also be known as mimic antibody, because of the characteristics similar to antibody. Anticalin has small molecular weight as genetic engineering antibody and offer some advantages over traditional antibody technology: First, lipocalin is more suitable to bind the small molecular; Second, the bag of recognition has large contact surface; Third, it is easier to manipulate by genetic engineering; Last, the Anticalin has high affinity and stability.Using the advantage of ribosome display build the Anticalin antibody library, we should to obtain the bilin binding protein （BBP） gene sequences and express BBP in E.coli at firs: the 12 long oligonucleotide primers were designed according to the natural BBP sequence and E.coli codon preference. The optimization sequence was amplified by overlapping PCR. After determined by sequence analysis, the BBP gene was cloned into pET-32a and pBV-220 to construct the recombinant expression vector pET-32a-BBP and pBV-220-BBP. The protein was expressed by induced as inclusion body, accounting for 40% and 65% of the total bacterial proteins. After guanidine denaturation and urea refolding, the fusion protein was successfully purified by Ni affinity chromatography. And over 98% of the fusion protein was obtained. Western blot proved that the purity protein was BBP protein.Using the synthesis BBP genes as a template. 16 oligonucleotide primers were designed which had random mutation to synthesis the antibody library. The all elements necessary used for ribosome display was amplified. Then, a 930bp library which encodes random 16 mutation used for Anticalin ribosome display was generated by using overlap PCR. The DNA library was identified by sequencing and in vitro transcription/translation.After transcription and translation of the Anticalin library in vitro, we got the antibody-ribosome-mRNA complex. The solid phage affinity method was used to screen anti-ampicillin antibody. AMP triple was dissociated by changing the Mg²⁺ concentration and the mRNA of mimic antibody was obtained. To screen the next round, the all elements necessary used for ribosome display was amplified again. Through several rounds of screening, highly specific anti-ampicillin gene was enriching. The antibody sequence could highly expressed in E. coli. The WB verified that the specific protein bands were the anti-ampicillin antibody. Because of the versatility of native antibody library, specific antibody can be selected for any kinds of antigen theoretically. This methods established the foundation that we can select specific antibodies of any antigens especially micromolecule haptens.