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Cloning, Expression And Function Of OPG And BmK ITa1 Gene

Posted on:2004-03-30Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z LiuFull Text:PDF
GTID:1100360122971015Subject:Biochemistry and Molecular Biology
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Two sections were included in this thesis.Section one: cloning, expression and function of OPG and its mutants OPG-280 and OPG-Fc. Osteoprotegerin (OPG), also termed Osteoclastogenesis inhibitory factor (OCIF), is a novel secreted protein involved in the regulation of bone resorption. It's reported that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis and hypercalcemic diseases. As a newly found protein, it is absorbing because of its reaseach significance and medical foreground. Presently the study of the structure-function relationship of OPG is mainly using the recombinant expression product from CHO cells, but its lower level limits its further research. So in this study we cloned OPG and its two mutants, and tried to express them in different expression system to find an ideal system for mass-produced biologically active recombinant OPG. The mature peptide of OPG is composed with 380 amino acid residues and present both as a monomer and a disulfide-linked homodimer, and the Cys379 is responsible for dimer formation of OPG. Previously we cloned a novel OPG cDNA (OPG-372, GenBank AF134187) by RT-PCR from cell line L02 established from normal Chinese human liver for the first time, sequence analysis showed that the OPG cDNA from L02 cells was a mutated OPG gene that had a nonsense mutation at the codon of Gln373 and OPG-372 isoform was 8 amino acid residues less than the OPG reported at the C terminal. The OPG-372 gene has been expressed with baculovirus system and proved that OPG-372 couldn't form homodimer.In order to compare OPG metabolize and biological stability between monomerand homodimer, OPG and its two mutants were constructed. The 3' fragment of OPG gene was amplified from peripheral blood lymphocyte (PBL) cells of normal Chinese, and the result of sequencing revealed that the genomic sequence was identical to the OPG reported. Recombinant with the OPG-372 and the full length OPG as reported was cloned. Considering that the multi-disulfide of Fc segment from human immunoglobulin gene IgG, the C-terminus of OPG-372 was fused with Fc and the lengthened mutant OPG-Fc was constructed. Additionally, according to the OPG characterization, we cut off the 100 amino acid residues in OPG-372 C-terminus and constructed a truncate mutant OPG-280 to research the minimum functional peptide of OPG. Then the OPG and its mutants OPG-280 and OPG-Fc were successfully expressed in E.coli, Tn-5B1-4 cell and Bombyx mori larvae. The OPG-280 in pET-28a vector of E.coli can be highly expressed and formed inclusion body. By renaturing and refolding, the purified OPG-280 was confirmed to be active by its obvious hypocalcemic effect in the normal mice. The blood was obtained 3h after injected and the serum calcium concentration was decreased 1.5mg/100ml (about 15%). Second, OPG and OPG-280 and OPG-Fc were inserted into the donor plasmid pFastBac1 and pFastBacHTb, and then expressed with high level in Tn-5B1-4 cells and homodimer of OPG-Fc was found according to the SDS-PAGE and Western blot analysis. All six recombinant products OPG,OPG-280,OPG-Fc,His-OPG,His-OPG-280 and His-OPG-Fc can decrease the serum calcium concentration by 10%, 11%, 15%, 8%, 10% and 14% respectively. Finally, OPG and OPG-280 and OPG-Fc were inserted into the baculovirus transfer vector pBacPAK8 and expressed in fifth instar B.mori larvae. Two homodimers of OPG and OPG-Fc were found and glycosilated OPG protein was observed. The hypocalcemic effect showed that the three products OPG, OPG-280 and OPG-Fc can decrease the serum calcium concentration by 19%, 14% and 17% respectively. Among above three expression systems, OPG-280 is present as monomer only. And OPG is present as homodimer only in B.mori larvae. While, except in E.coli, OPG-Fc can exist with two forms in Tn cells or B.mori larvae. These results conferthat the multi-disulfide of Fc can be helpful in forming OPG homodimer. Compared with OPG and its mutants from Tn cells hypocalc...
Keywords/Search Tags:Osteoprotegerin (OPG), scorpion neurotoxins BmK ITa1, cloning, mutant, recombinant expression, E.coli, baculovirus, Bombyx mori larvae, hypocalcemic effect, amidation, insecticidal activity
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