Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.
Electroporation ; Genetic Vectors ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; analysis ; biosynthesis ; genetics

Objective To set up a mouse model of spondyloarthritis,analyzethe clinical phenotype,radiographic and pathological features,and investigate the therapeutic effect of cysteine-rich 61 (CCN1) monoclonal antibody in spondyloarthritis mouse model.Methods Proteoglycan from bovine nasal septum was used for immunization of 14-16 week old female BALB/c mice.CCN1 monoclonal antibody 093G9 or control immunoglobulin (Ig)G were injected to the spondyloarthritis mice.The arthritis scores were analyzed by t test.Peripheral and axial joints disease development was assessed by Micro-CT and histology.Results Proteoglycan immunized mice began to develop peripheral arthritis in the 8th week.The peripheral arthritis score reached the peak (10.5±1.5) in the 11th week,with the inflammation and spur formation of the ankle and knee joint.We found infiltration of inflammation cells in intervertebral discs of the lumbar vertebrae and the caudal vertebrae.Chondrocyte proliferation couldbe seen in the meniscus of knee and lumbar intervertebral discs.In the 18th week,the intervertebral discsof thoracic vertebrae and the cervical vertebrae were also damaged.Abundant chondrocytesgathered in the intervertebra] discs.The inflammation and new bone for-marion of peripheral and axial joints were more severe in control IgG group than 093G9 group.The peripheral arthritis score in the 093G9 group decreased significantly after 2 treatments,[(2.8±1.3) vs (4.2±2.1),t=2.516,P＜0.05].The difference in arthritis scores between the two groups was the most significant after 8.treatments,[(2.0±2.0)vs (5.3±2.0),t=4.082,P＜0.01].Conclusion The mouse model of spondyloarthritissimulates human spondyloarthritis,including inflammation and new bone formation in p()gheral and axial joints.CCN1 monoclonal antibody can improve the inflammation and new bone formation inspondyloarthritis mouse model.