- A Recombinant Human GM-CSF Protein Expressed as an Inclusion form in Escherichia coli Stimulates Colony Formation and Cell Proliferation in vitro
-
Ah Young Lee , Jin-Kyoo Kim , Hye Kyung Chung , Eun Kyong Bae , Jung Suk Hwang , Chung Won Cho , Dong Seok Lee , Jae Yong Han , Choon-Taek Lee , Soon-
-
J. Microbiol. 2002;40(1):77-81.
-
-
-
 Abstract
 PDF
-
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematopoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator. In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His.Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-soluble form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.
- Molecular Cloning and Characterization of cDNA Encoding Immunoglobulin Heavy and Light chain Variable Regions from Four Chicken Monoclonal Antibodies Specific to Surface Antigens of Intestinal Parasite, Eimeria acervulina
-
Ki Duk Song , Jae Yong Han , Wongi Min , Hyun S. Lillehoj , Sung Won Kim , Jin-Kyoo Kim
-
J. Microbiol. 2001;39(1):49-55.
-
-
-
Abstract
PDF
-
We have developed four chicken hybridomas secreting monoclonal antibodies to induce a protective immune response against the chicken disease avian coccidiosis, caused by the intestinal parasite Eimeria acervulina. However, since the amount of antibodies secreted from these hybridomas is too low or sometimes they lost their ability to produce antibodies, the hybridoma method is not satisfactory in the production of large amounts of chicken monoclonal antibodies. To bypass these problems, we applied the antibody engineering technology using polymerase chain reaction. We cloned and determined the sequences of variable domains of the four chicken monoclonal antibodies, namely, 2-1, 5D11, 13C8 and 8C3. The sequences comparison to germline sequences showed that the gene conversion mechanism might contribute to developing diversification of heavy and l-light chains in chicken antibodies. Several pseudogene families regarded as donors in gene conversion were identified at each framework region and the complementarity determining region of l-light chains. In addition, as expected, numerous changes of nucleotide sequences such as nucleotide substitution, insertion and deletion were found predominantly in complementarity determining regions, which are likely to be somatic hypermutations as a result of affinity maturation in antibody-producing cells.
|
MSK
