Eukaryotic Expression Of Human Lipocalin-type Prostaglandin D Synthase In Pichia Pastoris And Preparation Of Its Monoclonal Antibody

Lipocalin-type prostaglandin D synthase (L-PGDS) , also known as b -trace protein( (3 -TP), is a unique member of the Lipocalin superfamily composed of various secretory lipophiilic ligand-carrier proteins with low molecular weight, because it was the first enzyme to be recognized in this superfamily. L-PGDS is localized mainly in the central nervous system and male genital organs of various mammls and is secreted into various body fluids, such as the cerebrospinal fluid, serum, seminal plasma, urine and aminotic fluid. L-PGDS is bifunctional, acting as a PGD2-producing enzyme and as a potential transporter of hydrophobic molecules. L-PGDS appears to be involved in several functions in vivo including induction of sleep, temperature regulation, nocieption and modulation of odour. L-PGDS is also closely associated with fertility, maybe playing an important role in the development and maturation of sperm. Studies indicate that monitoring the concentrations of L-PGDS in the body fluids are useful for the diagnosis of several neurological disorders, dysfunction of sperm formation, and cardiovascular and renal diseases. Therefore there is a need to acquire large amounts of purified L-PGDS and its antibodies, not only for the establishment of an immunological method to detect L-PGDS, but also for exploring its function in male reproductive system and relation with male infertility.In the first trial, human testis L-PGDS gene coding region was amplified from plasmid pGEX-2T/htL-PGDS by PCR with a deletion of the signal peptide sequence and cloned into pGEM-T vector. The sequence of the amplified DNA fragment was identical to that of human testis L-PGDS cDNA previously reported.Digestion of pGEM-T/htL-PGDS with EcoR I and Not I released the 0.55kb L-PGDS fragment which was thereafter subcloned into the same restriction sites of the pPIC9 yeast expression vector. This construct was designated pPIC9/htL-PGDS.In the second chapter, pPIC9/htL-PGDS was linearized with Bgl II followed by transformation of Pichia Pastoris GS115 with electroporation. After PCR analysis of Pichia intergrants, methanol was used to induce the expression of the his-tag protein. One recombinant clone was found to express L-PGDS with a molecular mass of 27 000, which was identical to that of native L-PGDS. The band at Mr 27 000 was about 18% of the total proteins in the culture supernant, and the expression level could be as high as 27mg/L.In the third part, the recombinant L-PGDS purified with Ni-NTA resin showed considerable heterogeneity with a major protein band at about MT 27 000 and two minor bands at about 24 000 ad 21 000. According to the results of glycosylation analysis, we speculated that the three protein bands were corresponded to the di-(27 kDa), mono-(24 kDa), and non-glycosilated(21 kDa) isoforms respectively. After forming the complex with purified L-PGDS, the UV spectra of all-trans retinoic acid were red-shifed approximately 30nm. In the immunoblotting test with L-PGDS polyclonal antibody, a highly specific 27 kDa band was observed.In the last chapter, the above purified recombinant L-PGDS was used as an antigen to immunize BALB/c mice, and then cell fusion was performed with 50% PEG. After limiting dilution, the hybridoma cells were inoculated to BALB/c mouse abdominal cavity to generate ascitic fluid-type L-PGDS McAb. One strain of IgG1 L-PGDS McAb was harvested, and the titer was 1:55-1:57. Western blotting of human seminal plasma samples probed with L-PGDS McAb was immunoreactive at 27 kDa.