摘要：

为探究神经生长因子(NGF)的基因功能,明确其在草原红牛不同组织中的表达及其对牛骨骼肌卫星细胞(bSMSCs)成肌分化的影响,采集草原红牛各组织,克隆并获得 NGF基因完整 CDS 区,并进行生物信息学及组织表达分析。从新生牛犊后肢的深层组织中分离bSMSCs,免疫荧光染色检测bSMSCs特异性标志物Pax7,Desmin和MyoD1的表达,并利用血清撤离法在体外使用2%的马血清诱导bSMSCs成肌分化,并检测了在分化的不同时期NGF基因mRNA和蛋白的表达规律。结果表明,草原红牛NGF基因 CDS 区长度为 726 bp,编码 241 个氨基酸;其氨基酸序列与家鼠、红鹿及绵羊位于一个分支,同源性较高,亲缘关系较近。NGF 蛋白分子质量约为26.67 ku,分子式为C₁₁₇₉H₁₈₆₁N₃₄₉O₃₃₉S₁₀,理论等电点为 9.72,半衰期为 30 h,NGF 蛋白为水溶性蛋白;NGF蛋白存在信号肽,是分泌蛋白;共存在 42 个潜在的磷酸化位点及3 个N-糖基化位点。NGF 蛋白二级结构由 α-螺旋、β-折叠、β-转角和无规则卷曲组成,并与三级结构预测结果一致。NGF基因在草原红牛不同组织中的表达量存在显著差异,在肝脏中最高,其次为肾脏和瘤。NGF基因在bSMSCs成肌分化过程中的表达是持续上调的,并在分化第7天呈显著性差异表达。成功克隆草原红牛NGF基因CDS区,并建立了NGF基因的组织表达谱,明确了NGF基因在bSMSCs成肌分化过程中的表达规律。

关键词: 草原红牛, NGF基因, 克隆, 组织表达差异, 生物信息学, 成肌分化

Abstract:

In order to explore the gene function of nerve growth factor(NGF),and to clarify the expression differences of NGF in different tissues of Red Steppe cattle and its influence on the myogenic differentiation of bovine skeletal muscle satellite cells(bSMSCs),collect various tissues of the Red Steppe cattle,clone and obtain the complete CDS region of the NGF gene,and then conduct bioinformatics and tissue expression analysis on it.bSMSCs were isolated from the deep tissues of the hind limbs of neonatal calves.The expression of Pax7,Desmin and MyoD1,the specific markers of bSMSCs,was detected by immunofluorescence staining.In vitro,2% horse serum was used to induce myogenic differentiation of bSMSCs by serum deprivation method,and the expression patterns of NGF mRNA and protein at different stages of differentiation were detected.The results showed that the length of the CDS region of bovine NGF gene was 726 bp,encoding 241 amino acids.The amino acid sequence of Red Steppe NGF gene is located in a branch of house mouse,red deer and sheep,with high homology and close kinship.The molecular weight of NGF protein was about 26.67 ku,the molecular formula was C₁₁₇₉H₁₈₆₁N₃₄₉O₃₃₉S₁₀,the theoretical isoelectric point was 9.72,the half-life was 30 h,and the NGF protein was water soluble.NGF protein had signal peptide and was secreted protein.There were 42 potential phosphorylation sites.There were three potential sites for N-glycosylation.The secondary structure of NGF protein was composed of α-helix,β-sheet,β-turn and random coil,and the results of tertiary structure prediction were consistent with those of NGF protein.There were significant differences in the expression level of NGF gene in different tissues,with the highest expression level in liver,followed by kidney and tumour.The expression of the NGF gene was continuously upregulated during the myogenic differentiation process of bSMSCs,and there was a significant difference on the 7th day.This study successfully cloned the CDS region sequence of the NGF gene in the grassland,and established the tissue expression profile of the NGF gene in the Red Steppe cattle.The expression pattern of the NGF gene during the myogenic differentiation process of bSMSCs was clarified.

Key words: Red Steppe cattle, NGF gene, Clone, Differentiation expression, Bioinformatics, Myogenic differentiation

中图分类号: 

• S823

• Q786基因的表达