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绵羊精子赤道膜蛋白片段1(SPESP1)的克隆、原核表达及纯化

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.023, PP. 155-160

Keywords: 绵羊,SPESP1,cDNA克隆,原核表达,蛋白纯化

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Abstract:

旨在克隆绵羊Spesp1cDNA并表达,得到纯化的GST-SPESP1融合蛋白。以绵羊睾丸组织总cDNA为模板,根据GenBank公布的绵羊基因组序列设计引物,PCR扩增得到Spesp1cDNA,构建原核表达重组载体pGEX-Spesp1,将其转化到E.coliBL21中表达,并优化其表达条件,利用SDS-PAGE切胶纯化法得到纯化的融合蛋白,用Westernblot鉴定所得融合蛋白。结果表明,经测序,克隆得到的Spesp1cDNA序列与GenBank中预测的cDNA序列对比有两个碱基不同,并造成一个氨基酸的差异。在E.coliBL21中成功表达重组融合蛋白,其最优表达条件为:37℃、4h、0.005%IPTG终浓度,SDS-PAGE和Westernblotting中,融合蛋白位于约64kD处并可以被抗GST和抗羊SPESP1的抗体识别,与预计相符,表明融合蛋白成功表达。成功纯化得到GST-SPESP1融合蛋白,为研究SPESP1在精卵细胞膜融合中的功能奠定了基础。

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