Construction Of Channel Catfish Naive Fab Fragment Phage Display Antibody Libraries

The antibody engineering technology has a revolutionary advance by using phage antibody libraries. It provides a powerful method to produce humanizd monoclonal antibodies and could obtain antibodies with higher affinity. The Fab phage antibody libraries of Channel catfish were constructed, because the biochemical information on structure and function of channel catfish immunogloblins had been known and there were no reports on the phage antibody library in aquaculture. The experiment results as following:1 Primer designIn the catfish VH gene segments are organized into 13 different VH families for which we designed a pair of primers. Channel catfish has two classes of light chain (designated F and G) which have a high degree of sequence similarity, so we designed two pairs of primers. There were restriction enzymes sites on both sides of these primers. The 5'-primers were corresponding to regions within the leader sequence or N-terminal FR region and 3'-primers were corresponding to the CH1 or CL regions extremity.2 Construction of the light chain libraryTotal RNA was extracted from the peripheral blood mononuclear cells (PBMCs) of 50 healthy channel catfish and then used to synthesize first strand cDNA. Heavy chain Fd fragments and light chain genes were amplified by a group of specific primers. The amplified products were purified and cloned into pMD18-T simple vector to get the recombinant vector pT-Fd, pT-L which were degested with restriction enzymes of SacI/XbaI or SpeI/XhoI and purified again in order to get heavy chain Fd and light chain genes. The second purifed products were inserted into the phagemid p3MH and then electro-transformed into competent E.coli XL1-Blue cells to construct the light chain library p3MH-L. The library members could be accounted by clones in agar medium flat. After several times of electroporations, the capacity of the light chain antibody library was 5.10×10~7. We selected 20 single colonies randomly which were amplified with primers of light chain and the bacilli PCR products showed that 18 fragments estimated to be 650bp in length were excised. The cloning efficiencies of light chain library were 90%. After degested with restriction enzymes of SacI/XbaI, the plasmid p3MH-L can be seen the insert size of about 650bp fragment which meaned that the light chain library was successfully constructed.3 Construction of the phage antibody libraryThe phagemid vector p3MH-L was incised by endonucleases SpeI/XhoI and big segments (about 4.1kb) were collecd. The big segments and the second purifed heavy chain Fd fragments were conjugated by T4 DNA ligase and electro-transformed into competent E.coli XL1-Blue cells to construct Fab phage antibody library. We calculated its capacity and recombine rate just following the way of light chain library. After 4 times of electroporations, the results showed that the Fab phage antibody library contained 5.67×10~7 different clones and the recombine rate was 90%. The cutting of enzymes showed that there were heavy chain Fd fragment and light chain genes in the phagemid and we have successfully constructed a channel catfish na(?)ve Fab phage antibody library.4 Analyzing the diversity of the antibody libraryAfter construction of Fab phage antibody library, single colonies were randomly selected in order to determin heavy chain Fd fragment and light chain genes DNA sequences. Multiple sequence alignments indicated that heavy chain Fd fragment and light chain gene structure was entire, antibody diversity was fine.