Expression Of Anti-Human CD25 Genetic Engineering Antibody And Immunotoxin In Pichia Pastoris

ObjectivesCD25 was highly expressed on the surface of activated T cells, resting T cells hardly expressed CD25, which provide the scientific basis to treat diseases mediated by activated T cells such as T cell lymphoma, autoimmune disease and transplant rejection.Based on Daclizumab (humanized anti-CD25 monoclonal antibody), we constructed the recombinant expression vectors pPICZalpha A-biscfv (Daclizumab) and pPICZalpha A-dmDT390-biscfv (Daclizumab). Biscfv (Daclizumab) and dmDT390-biscfv (Daclizumab) expressed by Pichia pastoris may provide new drugs therapy for lymphoma, transplant rejection and autoimmune diseases.MethodsSynthesis of target gene and construction of expression vector:using DNAWorks software to obtain primers with complementary oligonucleotide terminus.By assembly PCR method, we synthesized of four short gene fragments named scfv (1), scfv (2), scfv (3) and dmDT390 and connected them to the pMD19-T vector. After sequencing, four correct fragments by enzyme digestion were cloned to Pichia pastoris expression vector pPICZalpha A.Expression of recombinant protein in Pichia pastoris:expression vectors linearized transformed into Pichia pastoris GS115 by lithium chloride transformation method. Positive clones were screened on YPDS plate containing Zeocin.BMGY and BMMY were used for expressing recombinant proteins.SDS-PAGE and Western blot:After SDS-PAGE,the protein were transferred to polyvinylidene fluoride (PVDF),mouse anti-histidine tail antibody was used as the first antibody, and Horseradish peroxidase labeled Goat anti-mouse IgG antibody as the second antibody.Finally,the target proteins were visualized using chemiluminescence detection system.Protein purification:the proteins were purfied by Ni-Sepharose 6 fast flow resin, and the purified proteins were detected by high performance liquid chromatography (HPLC).Results1. We designed 24 oligonucleotide primers to synthesize scfv (1), scfv (2), scfv (3) and 44 oligonucleotide primers to synthesize dmDT390.The four gene fragments were synthesized by assembly PCR and cloned to pMD19-T vector.After sequencing, the correct scfv (1), scfv (2), scfv (3) and dmDT390 gene fragments were digested by enzyme and finally cloned to the vector pPICZalpha A to form the expression vectors pPICZalpha A-biscfv and pPICZalpha A-dmDT390-biscfv.2. We obtained stains having stably integration of the target genes by lithium chloride transformation method.3. The proteins expressed by recombinant Pichia pastoris integration of the biscfv genes were analyzed by SDS-PAGE (reducing).The biscfv was about 55kd that containing histidine tail verified by Western blot.4. The proteins expressed by recombinant Pichia pastoris integration of the dmDT390-biscfv genes were analyzed by SDS-PAGE (non-reducing).The dmDT390-biscfv was about 95kd that containing histidine tail verified by Western blot.5. The purity of protein detected by HPLC was more than 99%.ConclusionsRecombinant Pichia pastoris GS115 can stability expressed biscfv and dmDT390-biscfv proteins, and high purity proteins can be obtained by nickel column.