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

生物技术通报 ›› 2015, Vol. 31 ›› Issue (7): 155-160.doi: 10.13560/j.cnki.biotech.bull.1985.2015.07.023

绵羊精子赤道膜蛋白片段1(SPESP1)的克隆、原核表达及纯化

马媛媛, 程飞跃, 邢万金

(内蒙古大学生命科学学院，呼和浩特 010021)

收稿日期:2014-11-05 出版日期:2015-07-16 发布日期:2015-07-16
作者简介:马媛媛，女，硕士研究生，研究方向：分子遗传学；E-mail：myuan62@163.com
基金资助:
国家自然科学基金项目(31460600)，内蒙古高等学校科学研究项目(NJZZ14001)

Molecular Cloning，Prokaryotic Expression and Purification of Sheep Sperm Equatorial Segment Protein 1(SPESP1)

Ma Yuanyuan Cheng Feiyue Xing Wanjin

(School of Life Sciences，Inner Mongolia University，Hohhot 010021)

Received:2014-11-05 Published:2015-07-16 Online:2015-07-16

摘要/Abstract

摘要：

旨在克隆绵羊Spesp1 cDNA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Spesp1 cDNA，构建原核表达重组载体pGEX-Spesp1，将其转化到E.coli BL21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Western blot鉴定所得融合蛋白。结果表明，经测序，克隆得到的Spesp1 cDNA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.coli BL21中成功表达重组融合蛋白，其最优表达条件为：37℃、4 h、0.005% IPTG终浓度，SDS-PAGE和Western blotting中，融合蛋白位于约64 kD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。

关键词: 绵羊, SPESP1, cDNA克隆, 原核表达, 蛋白纯化

Abstract:

The purpose of this study is to clone sheep sperm equatorial segment protein 1 cDNA(SPESP1), express SPESP1 in prokaryotic cells. Firstly, SPESP1 cDNA was amplified with total cDNA of sheep testicular as template and the designed primers according to the sequence of sheep genome in GenBank. Then the amplified SPESP1 cDNA was cloned into the expression vector pGEX-4T1 to construct pGEX-SPESP1, which was transferred into Escherichia coli BL21 for expression and the conditions of expression were optimized. The purified fusion protein by SDS-PAGE gel-slicing was identified by Western blotting. By sequence alignment, two base pairs of cDNA sequence were different between the cloned SPESP1 and the predicted one in GenBank, which led to 1 amino acid varied. The expression of the recombinant fusion protein in E. coli BL21 succeeded under the optimal condition of 37℃ and 0.005% of IPTG for 4 h. SDS-PAGE and Western blotting analysis revealed that a protein at band of approximate 64 kD could be recognized by the antibody of anti-GST and anti-sheep-SPESP1, which was in accord with the prediction, and this implied that expression of fusion protein was achieved. In conclusion, successful gain of purified fusion protein GST-SPESP1 lays a foundation for the functional studies of SPESP1 in the membrane fusion of sperm and egg.

Key words: sheep, SPESP1, cDNA cloning, prokaryotic expression, protein purification

马媛媛, 程飞跃, 邢万金. 绵羊精子赤道膜蛋白片段1(SPESP1)的克隆、原核表达及纯化[J]. 生物技术通报, 2015, 31(7): 155-160.

Ma Yuanyuan, Cheng Feiyue, Xing Wanjin. Molecular Cloning，Prokaryotic Expression and Purification of Sheep Sperm Equatorial Segment Protein 1(SPESP1)[J]. Biotechnology Bulletin, 2015, 31(7): 155-160.

参考文献

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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

Molecular Cloning，Prokaryotic Expression and Purification of Sheep Sperm Equatorial Segment Protein 1(SPESP1)

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