| 4-1BB ligand (4-1BBL) is one member of tumor necrosis factor (TNF) family , which is mainly expressed on the B lymphocyte, antigen presenting cell (APC), and monocyte etc. It's receptor, 4-1BB, mainly expressed on activated T lymphocytes, including CD4+ and CD8+ T cells. The ligation of 4-1BB/4-1BBL plays a crucial role in initiation and regulation of the specific immune responses for cellular and humoral immunity.4-1BBL was identified in 1994. 4-1BBL protein is a type-II transmembrane glycoprotein, consisting of 255 amino acids(AA). Extracellular fragment from the 49th to 255th AA, nominated as soluble 4-1BBL, contains the domain by which 4-1BBL binds to 4-1BB receptor. Whether the soluble form of 4-1BBL possesses the same biological activity as the membrane 4-1BBL, which attracts our great attention. Therefore, we used the genetic engineering techniques to develope the soluble form of human 4-1BBL (rhs4-lBBL) and also the potential biological functions of this recombinant product was extensively investigated. Meanwhile, we constructed human 4-1BB transgenic cell line (L929-4-1BB) and explored its role and underlying mechanism on 4-1BBL reverse signal transduction.Part I. High efficiently expression and identification of human soluble 4-1BB Ligand in Pachia Pastoris GS115cDNA encoding the extracellular fragment of 4-1BBL was amplified from activated human B lymphocytes by using reverse transcript- polymerase chain reaction(RT-PCR) with a pair of artifical primers containing Hind III and EcoRI digestion sites respectively and then was cloned into PUCTm plasmid. After confirming the cDNA sequence, the soluble 4-1BBL(s4-1BBL) cDNA was recloned into the predigested yeast expression vector pPICZaA, which carries a factorsecretion signal sequence and with a Zeocin maker for antibiotic selection. The linearized pPICZaA-s4-1BBL plasmid with SacI digestion was introduced into Pichia Pastoris GS115 strain by eletroporation and integrated into the yeast chromosome. Postive recombinants were picked out by plating strains on YPD/Zeocin plates and sequentially were tested for S4--1BBL expression by culturing them in 2 ml of BMGY medium, followed in BMMY medium under the induction of 1% methanol. The target protein in supernatant of yeast culture was detected by 12%SDS-PAGE, western blotting. Through series of cloning selection with a gradually increased concentration of Zeocin, several high stable producing GS115/ pPICZaA-s4-lBBL strains were established. The rhs4-lBBL was quantitated by SmartspecTM3000 spectrophotometer . the maximum target protein yield of culture supernatant is 25 ug/ml with a 21Kda molecular weight at 72h after induced by methanol.For improving the producion of S4-1BBL protein, the automatically controled fermentator was used. Pichia Pastoris GS115 grows well in the defined basal salts medium for fermentation. The maximum cell density of 250g wet weight/L wasobtained.To purified the recombinant soluble 4-1BBL(rhs4-lBBL), the supernatant was harvested and dialyzed overnight at 40, against 20 mmol / L of tris-Hcl buffer, pH 8.0. The dialysate was loaded onto a Q-Sepharose column(PH=8.0) and FPLC was performed. The purity of the rhs4-1BBL after purified with FPLC reached to 95% of total protein in elution.Part II. the biological characteristics study of rhs4-1BBL 1. The costimulation effects of rhs4-1BBL on human peripheral blood T lymphocytesThe interaction of 4-1BB/4-1BBL exhibits a critical role in the regulation of immune response, including T cells proliferation, survival and cytokine secretion; In addition, the costimulatory signal of 4-1BBL to T cells is in most case independent of CD28 activations. Previous studies have shown that administration of agonistic 4-1BBmAb or 4-1BBL transgenic tumor cell can lead to generation of effectiveantitumor response. The soluble form of 4-1BBL is generated by the way of proteolytic cleavage mediated by one of more shedduse and has been likely showed to be a biological cytokine as membrane-bounding 4-1BBL to regulate 4-1B...
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