Production of STC-1 protein and monoclonal antibodies for therapeutic or clinical response monitoring in acute lymphoblastic leukemia

• Grant number: 15/08662-8
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Start date: December 01, 2015
• End date: August 31, 2016
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Luís Antonio Peroni
• Grantee: Luís Antonio Peroni
• Company: Rheabiotec Desenvolvimento, Produção e Comercialização de Produtos de Biotecnologia Ltda
• City: Campinas
• Associated researchers: Jörg Kobarg ; José Andrés Yunes
• Associated research grant(s): 16/16749-9 - Production of STC-1 protein and monoclonal antibodies for therapeutic or clinical response monitoring in acute lymphoblastic leukemia, AP.PIPE
• Associated scholarship(s): 15/25284-7 - Production of STC-1 protein and monoclonal antibodies for therapeutic or clinical response monitoring in Acute Lymphoblastic Leucemia, BP.PIPE

Abstract

The use of immunodiagnosis kits is one of the quickest and safest bets for early treatment of human diseases. The production of these kits entails, first of all, generating proteins or recombinant peptides of specific targets of the referred pathogens, with high levels of purity and mimickry of the native protein's structural and conformational characteristics, and in the second place, highly sensitive and specific monoclonal antibodies, in order to guarantee an early, efficient, quick and precise diagnosis. Acute Lymphoblastic Leukemia (ALL) is the most widespread cancer in childhood. The Stanniocalcin-1 (STC-1) protein was identified as a potential serum marker of ALL, useful to monitor clinical response to therapy. The present project focuses on cloning and expressing the STC-1 protein gene in E. coli bacteria and in an insect cell baculovirus system, as well as the expression and purification of recombinant proteins. In a second step, these proteins will be associated with the production of monoclonal antibodies in order to constitute an ELISA-based diagnostic kit, thus enabling an early diagnosis of the disease, with adequate sensitivity and reliability. The STC-1 protein gene (Genbank NM_003155) will be amplified from normal bone marrow stromal cells using specific primers, cloned in the pGEM vector and subcloned in the pET28a vector. The vectors will be transfected by electroporation in E. Coli prokaryote lineages and through the insect cell baculovirus expression system. Positive clones will be confirmed by PCR using universal primers for the prokaryote system and through GFP expression in the baculovirus system. The expression of STC-1 protein in the prokaryote system will be induced by IPTG and purified by nickel affinity chromatography. In the baculovirus system, the protein will be produced by infecting cells with the baculovirus culture and purified by chromatography in three steps: 1. Ion interchange; 2. Nickel affinity and 3. Molecular exclusion. All fractions will be assessed with SDS-PAGE (Laemmli, 1970), Indirect ELISA (Clark, et al. 1986) and Western blot (Towbin, 1979) in terms of purity, kDa weight and polihistidine tag (6-His-tag) reactivity, and with a rabbit-produced polyclonal, in order to trace the batches produced during the project. Results for this first phase will be the standardization of cloning and expression methodologies for these proteins, thus founding a platform for the commercial synthesis of these and future proteins, substituting imported similar and enabling Brazilian researchers access to locally-produced recombinant proteins, at better prices and delays than those practiced by representatives of imported brands. The second phase of the project, once the synthesis platform is established, will be focused on the scaling of production capacity and the development of monoclonal antibodies necessary for the ELISA Acute Lymphoblastic Leukemia early detection kit. (AU)