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目的 表达及纯化小鼠透明带结合蛋白sp3 8。方法 从BALB/C小鼠睾丸组织提取总RNA ,利用RT PCR技术扩增sp3 8基因编码区序列 ,并将其重组到含麦芽糖结合蛋白 (MBP)的融合蛋白原核表达载体 pMAL c2x中 ,在大肠杆菌中进行表达。结果 序列测定表明克隆获得的sp3 8全长与基因库所登录的序列一致。经IPTG诱导表达出的MBP sp3 8融合蛋白分子量约 88kD ,经Westernblot分析证实。经直链淀粉琼脂糖凝胶 (amyloseresin)亲和层析和SephadexG 10 0分子筛层析纯化后 ,MBP sp3 8纯度达 90 %。 结论 成功地获得sp3 8蛋白 ,为进一步研究其生物学性能和用于免疫避孕的可能性打下了基础
Objective To express and purify the zona pellucida - binding protein sp3 8 in mice. Methods Total RNA was extracted from the testis of BALB / c mice. The coding region of sp3 gene was amplified by RT-PCR and cloned into the prokaryotic expression vector pMAL c2x containing maltose binding protein (MBP) Bacillus expression. Results The sequence analysis showed that the full length of sp3 8 cloned was consistent with the sequence registered by gene bank. The molecular weight of MBP sp3 8 fusion protein induced by IPTG was about 88kD, confirmed by Western blot analysis. After purified by amyloseresin affinity chromatography and SephadexG 10 0 molecular sieve chromatography, the purity of MBP sp3 8 reached 90%. Conclusion The successful sp3 8 protein has laid the foundation for further study of its biological properties and possibility of immunocontraceptive