Identification And Characterization Of The HLA New Alleles And Preparation Of Their Phage-display Monoclonal Antibodies

The human Major Histocompatibility Complex(MHC)is located on the short arm of of the human chromosomes 6(6P21.31-21.33),consisting of a set of closely linked gene complex loci.It occupies 3.6Mb?4Mb,about 0.13%of the 3×109 bp human genome.The complex encodes human leucocyte antigen(HLA)and contains more than 150 protein coding gene loci,accounting for about 0.5%of the 32 000 expressed protein coding genes in the human genome.The MHC is the most complex and polymorphic genetic system known so far,and is also one of the most gene-dense regions in the human genome.Before the start of the Human Genome Project,the HLA gene complex was the best studied multigenic region in the human genome.As it was a particular focus for the Human Genome Project,the MHC became among the first multi gene regions of the human genome to be completely sequenced for the Human Genome Project in 1999.Subsequently,the genetic data has been updated continuously in this region.According to Shiina et al.by 2009,there were 253 loci have been identified in the total 3.78Mb region of the MHC,including 45 HLA genes and 208 non HLA genes.As is the most complex and polymorphic gene region known to humans,the MHC is highly polymorphic.The polymorphism is mainly manifested by the great amount of multiple alleles of the loci,and the distributions of the alleles are with ethnic and regional differences.The alleles' frequencies are different from distinct regions and different populations.As of 2017,there are 16755 HLA alleles has been reported in the IMGT(international ImMunoGeneTics project)/HLA Database.There are 12351 HLA class-I alleles,including 3913 HLA-A alleles,4765 HLA-B alleles,and 3510 HLA-C alleles.There are 4404 class II alleles,including 2058 HLA-DRBlalleles.The high polymorphism of HLA alleles is the representation of the complexity and diversity of the genetic background.It is result from the adaptability to the adverse environmental factors in the process of human evolution.It enables the human beings to have the more ability to resist the invasion of pathogens,which has important biological significance for the population's survival.In the past twenty years,with the development and of the molecular biological technology and the HLA sequencing typing technique,the discovery of HLA new alleles has accelerated.With the set up of the China Marrow Donor Program(CMDA)and the employment of the SBT(Sequencing Based Typing)technique,there are more and more HLA new alleles have been reported in the Chinese population by Chinese scholars.The phage antibody library technology is also called the phage-display antibody library,which is another important technological development in the history of antibody preparation,following the hybridoma monoclonal antibody technology.The phage antibody library technology is developed from the phage display technology.It is a new antibody preparation technology combining of the phage display and the antibody library technology.The phage antibody library technology has fundamentally changed the traditional hybridoma monoclonal antibody preparation process,by bypassing the hybridoma cell fusion and the clone selection process,which greatly shorten the preparation period.The proliferation cycle time can be shortened from months to weeks,which greatly improves the preparation efficiency.The antibody library capacity could expand to 106?109 clones.By this technology,the antibodies' gene sequence can be obtained and be further genetically engineered.Yet in the field of HLA monoclonal antibody preparation,the application of phage display antibody technology has not been reported.Since 2008,there are 33 HLA new alleles have been identified in our laboratory and all of them have been officially named by the WHO HLA factor Nomenclature Committee.In the first part of the thesis,we reported the identifications of the 6 HLA new alleles,analyzed the possible originations of the novel sequences and the assumptions of the molecular structure variation of the encoding proteins.In the second part,we firstly produced proteins of the extracellular domains of the molecules of the new allele:HLA-A*24:191,A*24:224,A*24:225,A*24:257,and HLA-A*24:02 using the prokaryotic expression system.Then we produced the monoclonal antibodies with the epitopes of the amino acid mutations encoded by the the new allele HLA-A*24:191,with the phage-display antibody library technology.The tentative studies of the preparations of the HLA monoclonal antibodies by the phage-display antibody library technology could be as the preliminary basis for the future preparation studies and also provide tools for further analysis and studies of the expressions and the molecular structures of the of these new alleles.Part 1Identification and Characteristics of the HLA New AllelesObjective:To identify and characterize six novel HLA allelic sequencesMethods:1.The routine HLA-A,B,DRB1 high resolution typing were used with HLA PCR-SBT(sequence-based typing)technique and the liquid phase fluorescent beads PCR-rSSO(reverse sequence-specific oligonucleotide probe)flow hybridization typing technique based on Luminex platform.2.The samples with abnormal results were typed again using the HLAssure SE SBT HLA Typing reagents which employs the single allele specific sequencing strategy.By this technology,the heterozygous alleles on the two haploids loci were sequenced separately on both directions as the single strand sequencing technique.3.By using SWISS-MODEL,Phyre2,RasMol and RCSB PDB Protein Workshop softwares,the protein structures encoded by the new alleles were modelled and analyzed.Results:1.The results of locus A of the sample 00333 were typed as A*02:06:01+A*68:01:02 by the Luminex fluorescent beads PCR-rSSO flow hybridization,but with 2 abnormal positive FP#022 and FN#072 false reaction probes.The SBT results showed that the most matching results were A*02:03:01+A*68:01:02,but with 2 nucleotides difference in exon 2 at position 98 where the result nucleotides changed from W(A+T)to A and at position 102 the result nucleotides changed from W(A+T)to Y(C+T).The repeated single allele-specific sequencing results were consistent with the initial SBT results and showed that the nucleotide differences are occurred on the A*02:03:01 allele at position 98 where T->A and at position 102 where A->C,which changed the codon 9 from TTC to TAC resulting in an amino acid exchange(F9?T)and the codon 10 from ACA to ACC with no amino acid change.2.The locus of HLA-DRB1 of the sample 01513 were typed as DRB1*15:66+DRB1*14:05:01/02 by the Luminex liquid phase PCR-rSSO flow hybridization,but with abnormal FP#516/517 false positive reaction probes.The SBT results showed that the most matching results were DRB1*14:05:01?DRB1*15:66,but with 2 nucleotides mismatches in exon 2 at position 258 where the result nucleotides changed from Y(T+C)to T(T+T)and at position 261 the result nucleotides changed from T(T+T)to Y(C+T).The repeated single allele-specific sequencing showed that the DRB1*15:66 has got 2 nucleotide substitutions at position 258 where C->T and at position 261 where T->C,which changed the codon 57,58 from GAC GCT to GAT GCC.Both of the substitutions resulted in no coding changes.3.The SBT results of locus A of the sample 00791 were typed as A*24:02:01+A*24:02:01.But there is a nucleotide mismatches in exon 2 at position 215 with the result nucleotides changed from G(G+G)to R(A+G).The repeated single allele-specific sequencing showed that one A*24:02:01 allele has got a nucleotide substitution at position 215 where G->A which changed the codon 48 from CGG?CAG,resulting in an amino acid exchange(R48?Q).4.The SBT results of locus A of the sample 00059 were typed as A*24:02:01+A*33:03:01.But there is a nucleotide mismatches in exon 2 at position 178 with the result nucleotides changed from T(T+T)to Y(C+T).The repeated single allele-specific sequencing showed that the A*24:02:01 allele has got a nucleotide substitution at position 178 where T->C which changed the codon 36 from TTC to CTC,resulting in an amino acid exchange(F36?L).5.The SBT results of locus A of the sample 00290 were typed as Aa*24:198+A*33:03:01.But there is a nucleotide mismatches in exon 2 at position 155 with the result nucleotides changed from T(T+T)to W(A+T).The repeated single allele-specific sequencing showed that the A*24:191 allele has got a nucleotide substitution at position 155 where T->A which changed the codon 28 from GTG?GAG,resulting in an amino acid exchange(V28?E).6.The results of locus A of the sample 00550 were typed as A*24:02:15+A*02:01:01 by the Luminex liquid phase PCR-rSSO flow beads reverse hybridization,but with a abnormal positive FP#43 false positive reaction probes.The SBT results were typed as A*24:156 + A*02:01:01.But there is a nucleotide mismatches in exon 2 at position 256 with the result nucleotides changed from G(G+G)to S(C+G).The repeated single allele-specific sequencing showed that the A*24:156 allele has got a nucleotide substitution at position 256 where G->C which changed the codon 62 from GAG?CAG,resulting in an amino acid exchange(E62?Q).All of the above sequence of the novel allele was submitted to the NCBI GenBank,and the nucleotide sequence data have been released in the EMBL,GenBank and DDBJ Database under the accession number JN209961,HG797712,JQ899198,JQ924283,HG003642 and JN209958 respectively.The name HLA-A*02:355,DRB1*15:06:02,A*24:224,A*24:225,A*24:257 and A*24:191 has been officially assigned by the WHO HLA factors Nomenclature Committee respectively.Conclusion:There are six new HLA alleles were identified and conformed.The names of HLA-A*02:355(sample 00333),HLA-DRB1*15:06:02(sample 01513),HLA-A*24:224(sample 00791),HLA-A*24:225(sample 00059),HLA-A*24:257(00290 samples)and HLA-A*24:191(00550 samples)have been officially assigned by the WHO HLA factors Nomenclature Committee respectively.Part ?Preparation of Monoclonal Antibodies against the New Allele A*24:191 Coding Protein by Phage-Display Antibody Libraries Chapter 1,Prokaryotic Expressions and Purifications of the Extracellular Domains of the HLA-A*24 New AllelesObjective:To prepare and purify the soluble Extracellular Domain Proteins of the A*24 new alleles by prokaryotic expression system and Ni-NTA Resin column.Methods:1.The Prokaryotic Expressions were performed according to the A*24 new alleles'sequences.An A*24 Total Mutation Sequence was designed which contains all the variation nucleotide sequences and the A*24:02:01 sequence is set up as the standard A*24 protein sequence.2.The template gene fragment was amplified by overlapping extension PCR method.The pET-28a(+)vector was digested by Ncol and Xhol restriction enzymes.The target gene fragment was inserted into the vector by homologous recombination through the seamless cloning technique3.The vector was transfected into TOP 10 competent cells and the positive clones were identified by PCR and sequencing.4.The objective protein was expressed by Transetta DE3 competent cells after transfected by the vector.5.The inclusion bodies were disrupted by ultrasound and purified by Ni-NTA Resin column.6.The sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis(PAGE)was performed to analyzed and identify the harvested and purified proteins.Results:1.The results of template overlap extension PCRs and the positive clone PCR tests showed that the sizes of the PCR products were correct and up with the expected standards.2.The sequencing results of the positive clones showed that the sequence of the inserted gene fragment was identical with the target gene sequence.3.The results of SDS-PAGE and Western blots showed that the sizes of the expressed proteins are accurate and all the proteins are with high purity.These soluble proteins could be used for further immunization and monoclonal antibodies screening.Chapter 2,Preparation of Monoclonal Antibodies against the HLA-A*24:191 Coding Protein by Phage-Display Antibody LibrariesObjectiveTo prepare the monoclonal antibodies against A*24 new alleles by phage display antibody libraries techniqueMethods1.5 BALB/c mice were immunized by the synthetic peptides 4 times with the interval of 3/2/2 weeks respectively.The mice's serum was tested by ELISA 7 days after the 4th immunization.Then 3 days later the reinforcement of immunization was performed.2.At the 2nd day of the reinforcement of immunization,RNA extraction from mice spleen and RT PCR was performed to get cDNA.3.The light chain and heavy chain genes were amplified by 25 pairs of light chain gene primers and 50 pairs of heavy chain gene primers.4.The light chain genes' PCR products and pHD vectors was digested by ApaLI and AscI restriction endonucleases.The PCR products of the heavy chain genes were digested by Notl and Sfil restriction endonucleases.5.The light chain genes inserted into the plasmid vectors,and then transfected to SS320 cells after desalinization and purification.6.The light chain library was sequenced and its capacity was determined,and the plasmids of the light chain library were extracted after the identification.7.The plasmids of the light chain library were digested by Notl and Sfil endonucleases for enzymatic cleavage.The heavy chain gene digested by the same two endonucleases is then inserted into the light chain Library.8.The Fab antibody library was sequenced and its capacity was determined.The diversity of the library,as well as the integrity,the species and the germlines of antibodies' genes were analyzed.9.The Fab antibody library was recovered,and then it underwent inoculation and culture,helper phage infection,screening and expanding culture.10.Phage precipitation and pannings,salvage and amplification,ELISA screening and confirmation,induction of antibody expression of positive clone.11.Western blot test with the supernatant of bacterial lysate and the prokaryotic expressed protein of A*24:191.Results1.The Elisa results showed that the immunization titers of mouse 3 were up to quality.2.The electrophoresis showed that the quality of RNA extraction,the results of the light chain and heavy chain genes amplifications and the endonucleases digestions meet the requirements.3.The capacity of light chain library was 3.6×106 pfu,and the sequencing analysis showed that the correct rate of the antibody gene insertion was 86%(6/7).4.The capacity of Fab antibody library was 4.4×107 pfu,and the sequencing analysisshowed that the correct rate of the antibody gene insertion was 80%(16/20).Sequence analysis of the heavy chain genes showed that they were all murine antibody sequences.The germline analysis showed that 70%(7/10)of the antibody genes was distributed in the IGHV1 family.The integrity of the antibody genes and the library diversity should be greater than 80%.5.After the phage display antibody library salvage and panning,ELISA screening and confirmation,Western blot test showed that the monoclonal antibodies in the supernatant of bacterial lysate of one clone selected could combine the A*24:191 expressed protein with higher affinity.