Different Evolutional Selection Of A Combinatorial Phage Library Displaying Randomly-rearranged Binding Domains Of Ig-binding Molecules With Human And Goat IgG

Staphylococcal protein A and streptococcal protein G are the bacterial host antibodyspecifically binding bacterial immunoglobulin-binding protein （Immunoglobulin（Ig）-binding Proteins, ibps to） representatives molecules. The SpA and SpG bothcontains repeat composed of a plurality of sequences are highly homologous to thebinding domain of the antibody binding region, the structure of each single domainhaving completely the function of the binding of IgG.In this study, two random phage display library SpA and SPG single domain havebeen Constructed, using human IgG and goat IgG direct evolutional selection of acombinatorial phage library displaying randomly-rearranged various binding domainsof protein A and protein G, and show whether combinations of Ig-binding domains ofvarious IBPs could produce useful novel binding properties. The gene fragmentsencoding the A、B、C、E domains of SPA, the B2、B3domain of SPG and the A、B、D、E domains of SPA, the B2、B3domain of SPG were generated by PCR amplificationusing the primers, which introduced recognition site for Xbaâ…  in both ends and arandom linking sequence in3’ terminal，which encoded random linking peptidecontaining3amino acids. All fragments of these domains,were digested byXbaâ…  enzyme and ligated randomly with each other,before recombined into thephagemid vector pCANTAB5S.The recombinants, were transformed into E.coli DH5αextracted from E.coli DH5α,and re-transformed into E.coli TG1and rescued by thehelper phage M13K07then a phage-displayed random combinatorial library ofIg-binding,Mono-domains of SPA and SPG was established.Size distribution of eachround of screening were randomly selected clones were identified by PCR fragmentwas inserted into the detection, to evaluate the effectiveness of screening,All thesephage clones displaying more than one domain were picked to make sequence analyses.After the reunification of the result of the screening, the final preparation of the specificcombination of advantages molecules monoclonal phage, using phage ELISA method to compare with the binding properties of human and goat IgG.This study successfully constructed phage display spa A, B, C, E, the SPG B2, B3)（library C） and SpA （A, B, D, E）, the SPG （B2, B3）（library D） of sixsingle bindingdomain random combinatorial library.Random combinatorial library of single-domainlibrary C storage capacity for8.5×10⁶phage library titer was1.82×1012TU/Ml PCRidentification of positive fragment was inserted into the rate of78%（a domain portfolioaccounted for50%;2domainportfolio accounted for16.7%;3and three or moredomain portfolio accounted for8%）; library D capacity to8.1×10⁶phage library titerwas1.69×10¹²TU/Ml PCR identification of positive fragment was inserted into the rateof72%（a domain portfolio accounted for40%;2domain portfolio accounted for19.4%;3and3domain portfolio, accounting for9.5%）.Sequence analysis showed that thecombination of each single-domain case, the pros and cons to insert the connectingpeptide showed a random distribution of the library meet the in vitro evolutionscreening requirements.Filter last have the permutations molecular are not exist in thenative molecule SpA, the library C after human IgG induced by four-wheel filter, thefragment size is unified to2Domain sequencing analysis combined A-C, the library Cafter the sheep IgG the induction of six rounds of screening, the unity of the fragmentsize of2domain, sequencing combinations for the A-B3. The library D after humanIgG induced six screening, the unity of the fragment size3Domain sequencingcombinations for the E-B-B3, the library D after sheep IgG induced after six rounds ofscreening, the unity of the fragment size4Domain, combination for sequencinganalysis D-A-B3-B3.Phage ELISA further confirmed that the library of C is acombination of molecular A-C for human IgG binding capacity is higher than thecombination of molecules A-B3, a combination of molecular A-C for goat IgG bindingcapacity is less than the combination of molecules A-B3, fully consistent with thescreening results; however, the library D resulting composition Molecular D-A-B3-B3for human and goat IgG are higher than the combined molecular E-B-B3, does notshow the specificity of antibody binding. The research success in using human and goat IgG-induced bacterialimmunoglobulin-binding molecular single-domain random combination of phagelibrary to complete the in vitro evolution filter.we get four molecules with differentbinding strengths A-C, A-B3, E-B-B3, D-A-B3-B3, the only library C novelcombination of molecules have specific binding capacity with human and goat IgG.Thestudy could also provide new means for the further study of the bacterial the IBPstructure and function and the Fc portion of IgG binding molecules; and provides a newapplication prospects for IgG purification, preparation, detection.