The Natural Human Phage Antibody Library Construction And Initial Screening

By inserting the diverse genes from the variable domain of antibody into expression vector,expressing on the perface of phage,we get the collection of the diverse phage antibodies which is named the phage antibody library.Comparing to the polyclonal serum antibody and hybridoma technique,this phage antibody library based on the development of phage display technique has brought a large innovation to antibody engineering.It not only solves the problem from the preparation of the human antibody,but also makes the improvement of the performance of antibody has entered a new phase.We can use this technique to select some unkown structures with specific performance.With the improvement of construction techniques and screening methods, phage display antibody has been increasingly employed in many fields.It shows great advantages in production of high affinity antibody from human source,which could produce antibody without immunization procedure.Phage display antibody library can be divided into two types reference to the source of the antibody gene:combinatorial library,semi-synthesized library and natural library(immune repertoire and na(i|¨)ve library).Specific antibodies to a specific antigen with which immunized supplier could be acquired from immune repertoire.Antibodies acquired from immune repertoire have strong antigen-specificity and affinity. Non-immune library is ideal for the selection of specific antibodies against many antigens.Antibody genes are amplified from mRNA prepared from peripheral lympheral lyphocytes,spleen cells,bone marrow or tonsil B cells,which are inserted into phagemid vector to construct large naive library,it contains lots of phage display antibodies which can react with different antigens.Large naive phage display antibody library has many advantages such as antibody against self-antigens,non-immunogenic antigens and toxic antigens can be aquired;many antigens can be screened against one library;production of antibody in short time;high affinity antibody can be harvested.It also has many disadvantages such as low affinity antibody will be obtained from a small size library,long time of library construction,unknown immune background of donors, potential limited diversity of IgM library etc.Phage display library technology and Cre-Loxp recombinant system are used in this study to construct a large size of na(i|¨)ve antibody library which were screened by several antigens for the purpose of obtain specific human antibody.For the purpose of construction a high quality library,peripheral blood cells from 150 normal adults were collected for lymphocytes isolation and total RNA preparation,cDNA was synthesized using random oligo-dT primers and reverse transcriptase following standard protocols, antibody V genes were amplified with specific primers,Light chain genes were mixed according the natural ratio of antibody distribution(κ:60%λ:40%HVK1:19.2% HVK2:10.2%HVK3:30.6%HVL1:14.8%HVL2:13.2%HVL3:9.2%) then inserted into the vector pDF first to obtain a light chain library.A library was constructed by repeated electrotransfer,the insertion ratio of light chain was 100% among random picked clones.A primary library containing 8.8×10⁸ clones was constructed after heavy chain insertion according the natural ratio(HVH1:38% HVH2:34%HVH3:2%HVH4:26%).Primary library infected BS1365 which can secrete Cre enzyme inducing the genes to shuffle at 30℃at MOI＞100,recombinant library infected XL1-Blue at MOI＜1,the genotype and phenotype were unified.A library containing 9×10¹⁰ clones was harvested. Ten clones was randomly tested.Eight out of the ten clones(80%) has indicated the insertion of heavy chain and light chain,sequential analysis of randomly picked 96 clones showed that the antibody gene distribution is similar to the natural distribution, no sequence was the same and the length of CDR3 in heavy chain was between 4 to 20 amino acids,the diversity of the library is ideal for screening.Panning the library with cobra-venom and HCV NS4 antigens in high through-put was successful.Clones were random picked from the fourth round panning and expressed in E.coli XL1-Blue for picking antibody clones against two antigens.We have obtained 20 antibody clones which can react with anti-human IgG antibody,2 of them can react with antigens,1 clone can react with cobra-venom specifically,1 clones can react with HCV NS4,sequence analysis indicated that the type of light chain is VLⅡ,the heavy chain type is VH4.But the result of screening isn't satisfactory.It need improve the procedure and condition of the study to gain the antibody clones which have better specific performance.A large size of na(i|¨)ve antibody library has been constructed with phage display library technology and Cre-Loxp recombinant system,the quality of the library was analyzed by sequence and alignment,enrichment against two antigens was obvious,we have obtained 20 antibody clones which can react with anti-human IgG antibody by high through-put screening method which can be automated,one can react with cobra-venom specifically,another can react with HCV NS4 specifically.Quality of the library,screening method and condition should be adjusted for high affinity antibody selection.