The New Allele Hla-b ~ * 15:133 Identification Of The Heavy Chain Extracellular Polypeptide In The Prokaryotic Expression And Specificity Of Monoclonal Antibody Preparation

Part I: Identify a novel HLA‐B*15:133 allele in Chinese population Objective: To identify a novel HLA‐B*15:133 allele in Chinese population, the serological and generic investigation of family histories were performed for this new allele HLA‐B*15:133.Methods: During routine typing work, genomic DNAs of all samples'were extracted by salting‐out method and typed by PCR‐SSO and PCR‐SBT, an ambiguous result was found and identified a novel allele by DNA sequence analysis.Results: The Exon 3 nucleotide sequence of this novel allele is different from all other known alleles. It has one nucleotide substitution at position 419 from C to T (codon116 TCC‐>TTC), which results in an amino acid change from serine(S) to phenylalanine (F) in comparison to the most homologous allele B*15:18:01. However, serologic specificity remained B71(70) based on serological HLA typing. The results of family genetic and serological analysis indicated that the novel HLA‐B allele of the donor was inherited from his mother.Conclusion: A novel HLA allele was confirmed by the sequencing based typing (SBT) and it was officially designated as HLA‐B*15:133 by WHO Nomenclature Committee for Factors of the HLA System.Part II: Construction and identification of the prokaryotic expression vector pET 30a(+) containing human HLA‐B*15:133 Objective: The prokaryotic expression vector containing the extra‐membrane fragement of heavy chain of HLA‐B*15:133 was constructed and the expression of interesting protein was identified in E. coli.Methods: RT‐PCR was used to amplify the HLA‐B*15:133 cDNA from total RNA of leukocyte in peripheral blood. The extra‐membrane gene fragement of HLA‐B*15:133 (digested with BamHI and HindIII) was inserted into vector pET 30a(+). After identified the direction of insert, the expression vector pET 30a(+)‐B*15:133 was transfected into the BL21(DE3), and western‐blotting was used to identified the expression of the extra‐membrane petide of HLA‐B*15:133. Results: The expression vector pET 30a(+)‐B*15:133 was constructed successfμlly by using restriction endonucleases digestion and PCR. The expression of extra‐membrane polypeptide chain of HLA‐B*15:133 was determined by Western‐blotting.Conclusion: The expression vector pET 30a(+)‐B*15:133 can express the extra‐membrane peptide of HLA‐B*15:133 in BL21(DE3).Part III: Preparation and characterization of monoclonal antibody against new allele HLA‐B*15:133Objective: The monoclonal antibodies (mAb) against HLA‐B*15:133 were prepared and characterized.Methods: Based on the sequence of this new allele, three peptides were selected to conjugate with protein carrier KLH and BSA. The peptide‐KLH conjugation was used to immunize the Balb/c mice to generate hybridoma by using B lymphocyte hybridoma technique. The BSA conjugation was used for detecting and screening hybridoma by ELISA. The specificity of this mAb for extra‐membrane peptide of this new allele expressed by BL21(DE3) was tested by ELISA and Wetern‐blotting.Results: Six hybridoma cell lines against HLA‐B*15:133 were established which include 2 of IgG1/kappa, 2 of IgG2a/kappa, 1 of IgG3/kappa isotype and 1 of IgM /kappa isotype. Only one monoclonal antibody can recognized the expressed protein by BL21(DE3) which showed the positive bind in western blot.Conclusion: One hybridoma cell lines against HLA‐B*15:133 peptide with high specificity were generated which offers a useful tool for functional research on HLA‐B alleles in the future.