Study On Deer Velvet Antler Polypeptides And Analysis Of IGF-15′ End Regulatory Sequence

Deer velvet antler is the only mammalian organ capable of de novoregeneration and has been used as traditional oriental medicine. We extractedthe active components of fresh velvet antler—polypeptides which showed somebands below 1.7 kDa in SDS-PAGE, including IGF-1, EGF and NGF, withacetic acid solution(pH3.5)and alcohol. Quantitative analysis indicated thatpolypeptides were 1.78±0.23 mg/g of green weight by Coomassie brilliantblue G-250 technique, IGF-1 was 14.62±0.92 ng/g of green weight in acidextract and 352±58 pg/g of green weight in acid and alcohol extract byradioimmunoassay(RIA). Comparison of polypeptides, IGF-1, EGF andβ-NGF contents between the distal and proximal segments with RIA andELISA revealed that they are highly concentrated in antler tip(P<0.05).Mice were injected with polypeptides 1,4,8 mg/kg of body weight, aftersix days, the mean accumulative weight and daily weight gained inpolypeptides groups were higher than the control, but not significantly(P>0.05);The rosettes looping rates of the 1,4,8 mg/kg polypeptides groups were33.3%(P<0.05), 34.2%(P<0.05)and 39.6%(P<0.05) higher than thecontrol(injected with NS), respectively;OD value of haemolysin in serum of8 mg/kg polypeptides group was different significantly compared with thecontro(lP<0.01);The phagocytosis percentage of the 1,4,8 mg/kg polypeptidegroups were 22.1%(P<0.05), 25.8%(P<0.01), 26.5%(P<0.01)highersignificantly than the control, which indicated that the polypeptide has thefunction of immunological regulation. The superoxide dismutase(SOD)activities and malondialdehyde(MDA)contents in serum of old mice injectedwith 4,8 mg/kg polypeptides groups which increased by 51.1%, 59.2% anddecreased by 14.9%, 16.7% separately than the control group(P<0.05),showing the anti-lipid peroxidation function of polypeptides. In the weightswimming experiment, the swimming time of 8 mg/kg polypeptide group waslonger 21.67 mins than the control(P<0.05), revealed the anti-fatiguefunction of polypeptides. All the above data suggested that velvet antlerpolypeptides could enhance immunity, anti-fatigue and anti-aging in mice.To identify the molecular mechanism of highly expressed IGF-1 in antlertip, 5′ upstream sequence of IGF-1 gene was obtained by PCR amplification(CCS1,074, Genbank Accession No. DQ234,266). Homology comparisonamong goat, sheep, bovine and deer indicated that velvet antler IGF-1 5′sequence was different to others(with homology 96.8%). Bioinformaticanalysis of CCS1,074 predicted that CpG island was at 180～310 base,promoters may be at 142～192,238～288,874～924,1,359～1,409 base, 253transcription factor binding sites were found. HAND/HAND2_E12.01 andZF5F/ZF5.01 whose relationship with velvet antler growing was waiting forfurther studies were screened for differentially potential transcription factors ofvelvet antler. Combined with the known IGF-1 sequence and bioinformaticanalysis, we concluded that: In CCS1,074, <1～1,580 base was the 5′ end ofIGF-1;exon 1 and intron 1 localized in 1,581～1,966 base and 1,967～>2,087base separately;the amino residues at 1,903～1,966 base were characterizedsignal peptide.