[The cloning and expression of rat insulin-like growth factor I gene].
In this study an exons-connecting technique was used to amplify the complete DNA sequence encoding the mature peptide of IGF-I. Two pairs of primers were synthesized, primer a (Pa) and primer b (Pb) were used to amplify exon 2 coding fragment while primer c (Pc) and primer d (Pd) for amplifying exon 3 coding fragment. Pb was 40 bp, 18 bp at its 5' end was same with the anti-sense of exon 3's 5' end. Pc consisted of 41 bp with 22 bp at its 5' end consistent with the sense of the exon 2's 3' end. Genome DNA was extracted from rat liver. Using rat genome DNA as template PCR was performed with Pa, Pb and Pc, Pd as primers respectively. Two kinds of PCR products were obtained. One was 90 bp corresponding with the exon 2, another was 160 bp corresponding with the exon 3. Another PCR was done with these two PCR products used as not only template but also primers for each other. A 210 bp DNA fragment was produced, which encodes the mature peptide of rat IGF-I. Firstly the gene was cloned into plasmid pUC18. Then the recombinant plasmid pUC-IGF-I was digested with restriction endonuclease BamHI and EcoRI, plasmid pGEX-3X was digested with the same enzyme. IGF-I gene was linked into pGEX-3X and expressing plasmid pGEX-IGF-I was constructed. Plasmid pGEX-IGF-I was transformed into E. coli DH5 alpha and induced to express by IPTG. Expressed protein was analysized by SDS-PAGE. A fusion protein of GST-IGFI about 32.5 kDa could be observed. Western blot was performed with goat anti-human IGF-I IgG as primary antibody and donkey anti-goat IgG HRP as secondary antibody. Its result confirmed that the fusion protein really containing IGF-I. GST-IGFI was purified by affinity-chromatography and carrier-protein GST was cut off by Factor Xa. The bioactivity of purified IGF-I was detected with cultured NIH3T3 cell. The recombinant rat IGF-I can promote cell proliferation, shown by the elevation of 3H-TdR incorporation.
Liang
Dong-Chun
DC
Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China. liangdongchun1225@263.net
Guo
Gang
G
Zuo
Ai-Jun
AJ
Zhang
Jing-Yu
JY
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D011993:Recombinant Fusion Proteins; D014316:Tritium; D007334:Insulin-Like Growth Factor I; D004247:DNA; D005982:Glutathione Transferase; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D013936:Thymidine