| IBPs is a crucial pathogenic factor, which is produced by various pathogens and combined with Ig in the form of non-antigen. It contains Sp A, Group C and Group G Streptococcus, Sp G and part of Pp L etc. According to the findings, this kind of molecule consists of binding domains with many highly homologous and sequence. Each single binding domain, which has the totally same binding character, forms of Ig’s binding domains. Because of the unique binding character, this molecule is widely used in antibody purification and identification, adsorption treatment and the pathogen specificity detection.Using the in vitro molecular evolution to get Acl that is recombined by Sp A A Ig binding domains and Sp A C Ig binding domains. After recombining of each single binding domain between Sp A E,D,A,B,C and SPG B2,B3.This molecular is of the new high binding character IBPS which is sifted through Phage display technology. So, Whether can we obtain higher binding character domains from using antibody evolution platform ? This research is work on that random Fc binding sites amino acid mutagenesis on Sp A A,C domains, construction of Phage display library A 127CI20.Using human Ig G autoinducer to sift in vitro affinity from two libraries expect to get mutants enhanced human Ig G combining capacity. So that we can get new ways of IBPs structures and functions.This study will be divided into the following two parts:Part Ⅰ:The construction of Sp A single domain mutant combinatorial libraryBuilt to meet the requirements of in vitro screening Sp A single domain structure A library in 29, 30 amino acid mutations(A1) Sp A single domain structure in 36, 37 C point mutations library(C1) and insert 3 amino acids after 37 random peptide(AI37) in the Sp A single domain structure A、insert 3 random peptide(CI20) connection after 20 C in the Sp A single domain structure. Name the phage library which is combined by A1 and C1 randomly as A1C1.The Capacity of these two libraries are 3.0×106 and 2.0×106 respectively. The drop degrees are 2.3×1012 and 2.1×1012(TU/ml) respectively. Each of the two libraries has affinity screened four or five times, then fully evolved. Deal with the mutational fragment by Xba I, Connect the fixed point mutation A1、C1 that has dealt with the p CANTAB5 S that has inserted mutation AI37、CI20 and has dealt with by CIAP digesting in the same way. In the conversion of E.coli TG1 with our fabrication, via M13K07 salvation, I get 2 AC mutant library. OF the all 16 single domain structures that have inserted, the Ratio of the number A1:C1: AI37: CI20 is 5:3:4:4, with randomness. In the insert segments of single domain structure, A C also appeared of the same proportion, which shows the two library’s mutational base type has a good randomness. The above results show that the two structured AC mutant combination phage library can meet the requirements for screening in vitro evolution. Part Ⅱ: Evolutional selection of the mutant phage library with human Ig GApplication of human Ig G as bait, through the "adsorption"- "wash-out"- "amplification" repeating cycle, for the construction of good library respectively has been screened in vitro molecular evolution, through screening four or five times, the complete disappearance of library hollow phage, the high gather of the multiple structure domain that all show the library has evolved fully. In the library which has screened, Squelchy test the appeared positive clones and the functional verification of Phage-Elisa. Summary: 1 This study successfully build Sp A AC single domain structure combined mutant phage library.2 Application of human Ig G as bait and molecular evolution in vitro screening the phage library to gain some kind combination of molecules AQ29K30AI29I30and AI37-TQSA which does not exist in the natural molecular. 3 The binding combination features of AQ29K30AI29I30 and AI37-TQSA functional verification by Phage Elisa... |
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