Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neurites outgrowth
Chinese Medical Journal 2002; 115(9): 1352-1357，-0001，（）：
observed it s activity in stimulating neurite outgrowth in vitro. Methods cDNA encoding mature human HBNF was amplified from total RNA isolated from an 182week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into p PIC9K, a shuttle expression vector for yea stsystem. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichiastrain GS115 by electroporation. His+ transformants were selected on minimal dextrose medium (MD) plates which were histidine free1 His+ yeast recombinants with multi-copy insert s were screened in vivo by their resistance to G4181 PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome1 Secreted expre ssion of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe it s ability to stimulate neurite outgrowth. Results In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the α-factor, and it s open reading frame was in frame with the α-factor signal sequence in p PIC9K1 SDS2PAGE showed that the molecular weight of the induced expression product was about 18kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells. Conclusion Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and it s product possesses the biological activity to promote neurite outgrowth.
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