Construction And Expression Of Genetically Engineered Antibodies To Human Lipocalin-type Prostaglandin D Synthase

Lipocalin-type prostaglandin D synthase (L-PGDS), also known as β -trace protein( β -TP), is a unique member of the Lipocalin superfamily. L-PGDS is mainly localized in the central nervous system and male genital organs of various mammals, 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. Furthermore, 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 is useful for the diagnosis of several neurological disorders, dysfunction of sperm formation, breast and ovarian cancers.To establish an immunological method to detect L-PGDS, and to explore its relationship to male infertility, human testis L-PGDS gene was expressed in Pichia Pastoris, and the polyclonal and monoclonal antibodies to L-PGDS were also obtained in our previous studies. But the polyclonal antibody in fact was multiple antibodies mixture, and the monoclonal antibody was heterogenous, that restricted their functional studies and clinical applications. The ideal monoclonal antibody was ought to origin from human, but which could not to come true in nowdays, because of hybridoma technique , affinity and so on. So preparing genetically engineered antibodies is an effective method to solve the problem. In this study, we wanted to construct and express ScFv and human-mouse chimeric antibody to L-PGDS, which would lay a foundation to its clinical investigations and applications.In the first chapter, the variable region genes of L-PGDS McAb were amplified by RT-PCR from hybridoma cells, using 5' degenerate primers of leader sequences. After sequenced, one VH and two Vk were obtained. But one of the V_κ was anaberrant Vk transcript, derived from myeloma cell line. The other VH and Vk were functional genes, for they were highly homologous to the variable region genes of murine immunoglobulin.In the second chapter, the variable region genes of L-PGDS McAb were assembled to a whole ScFv gene by SOE PCR, using a Linker [(Gly)4Ser]3. Then we constucted ScFv to expression vector pET-28a(+), and expressed it in E.Coli BL21. As a result, many soluble ScFv were demonstrated by SDS-PAGE, which was expressed under 25 °C or 28 °C and BL21 was abducted by 0.02 mM IPTG. Then we used Ni-NTA affinity chromatography to purify these soluble proteins, and the concentration reached to 2 mg/100 ml. The purified ScFv was proved to have L-PGDS antigen binding activity and specificity by ELISA and immunofluorescence competitive test.In the last chapter, making use of enzymatic digestions, the VH and Vk were joined to expressing vectors and constructed recombinant plasmids pAG4622/VL and pAH4604/VH. Then we transfected the plasmids one by one into the same Sp2/0 cells by DOTAP Reagent, and used mycophenolic acid and histidinol to select transfecants. As results, the Sp2/0 cells expressed pAG4622/VL steadily, that was confirmed by ELISA and RT-PCR. But the pAH4604/VH transfecants were not acquired. In a word, human-mouse chimeric light chain to L-PGDS was expressed successfully, which laid a foundation to obtain its whole chimeric antibodies further.