Expression Of Human SolubleCD40 Ligand In Pichia Pastoris And Study On Its Biological Functions

ABSTRACT CD4O ligand (CD4OL) is one member of tumor necrosis factor (TNF) family, which is mainly expressed on the activated CD4~ T lymphocytes. Occupation of CD4O receptor exsiting on B lymphocyte, antigen presenting cell (APC), and monocyte etc by CD4OL plays a crucial role in initiation and regulation of the specific immune responses including humoral and cellular immunity. The importance of CD4O and CD4OL interaction system is fully presented in patients with X-chromosome-linIed hyper-IgM syndrome and in mice with CD4O or CD4OL gene knockout. CD4OL protein is a type-il transmembrane glycoprotein, consisting of 261 amino acids(AA). Extracellular fragment from the 45th to 261 ~?aa, nominated as soluble CD4OL, contains the domain by which CD4OL binds to CD4O receptor. Whether the soluble form of CD4OL possesses the same biological roles as the membrane CD4OL gives rise to our interest and whether there are possibilities for soluble form CD4OL to be used to develop dendritic cell (DC), a most powerfi.il antigen presenting cell, in vivo, and in clinical tumor immune therapy attracts our great attention. However, it is yet no commercial product of recombinant human CD4OL to supply untill today both in china and abroad. Therefore, we are first in China to use the genetic engineering techniques to develop the soluble form of human CD4OL and also the potential biological functions of this recombinant 12 rhsCD4OL ~ product is extensively investigated. Part I. High Efficiently Expression of Human Soluble CD4O Ligand in Pachia Pastoris GSI 15 cDNA encoding the extracellular fragment of CD4OL was amplified from activated human CD4i T lymphocytes by using reverse transcript- polymerase chain reaction(RT-PCR) with a pair of artifical primers containing XhoI and XbaI digestion sites respectively and then was cloned into pSK plasmid. After confirming the cDNA sequence, the soluble CD4OL(sCD4OL) cDNA was recloned into the predigested yeast expression vector pPICZcLA, which carries a factor secretion signal sequence and with a Zeocin maker for antibiotic selection. The linearized pPICZaA-sCD4OL vector with Sad digestion was introduced into Pichia Pastorzs GS 115 strain by eletroporation and integrated into the yeast chromosome at AOX locus. Postive recombinants were picked out by plating strains on YPD/Zeocin plates and sequentially were tested for sCD4OL expression by culturing them in 2 ml of BMGY medium, followed in BMMY under the induction of 1% methanol. The target protein in supernatant of yeast culture was detected by 12% SDS-PAGE, ELISA and Western Blotting. A maximum target protein yield of 50 mg /L culture supematant was gotten, with a 3 OKda molecular weight. For improving the producion of sCD4OL protein, the automatically controled fermentator was used. Pichia Pastorzs GS1]5 grows well in the defined basal salts medium for fermentation. The maximum cell density of 280 g wet weight/L was obtained and the concentration of sCD4OL in fermentation medium is about two times of that in shaking flask. To purified the recombinant soluble CD4OL(rhsCD4OL), the supematant was harvested and dialyzed overnight at 40C against 20 mmol I L of phosphate 13 rhsCD4OL ~...