牦牛重组肌肉生长抑制素的原核表达与免疫原性分析。

Prokaryotic expression and immunogenicity analysis of yak recombinant myostatin.

机构信息

College of Life Science and Technology, Southwest University for Nationalities, Chengdu, China.

出版信息

Anim Biotechnol. 2012;23(4):253-60. doi: 10.1080/10495398.2012.722157.

DOI:10.1080/10495398.2012.722157

Abstract

Myostatin (MSTN) is a negative regulator of skeletal muscle growth. The objective of the present study was to express yak (Bos grunniens) recombinant MSTN protein in E. coli and study its characteristics of immunogenicity. cDNA encoding yak MSTN mature peptide was amplified by reverse-transcription PCR, and cloned into pET28a(+) vector and expressed in E. coli. The expressed recombinant MSTN was purified by affinity chromatography and used to prepare rabbit anti yak MSTN antibody. The results showed that yak MSTN mature peptide gene contained 330 bp nucleotides coding 109 amino acids. Content of the target protein accounted for 21% of the total expression products when MSTN-pET28a(+)-BL21(DE3) bacterium was incubated in LB medium with 0.1 mM IPTG for 6 hours. The molecular weight of the purified yak MSTN recombinant protein was 16.5 kDa, exhibiting excellent immunogenicity as shown by ELISA. The obtained recombinant MSTN of yak is suitable for further analysis of yak MSTN functions.

摘要

肌肉生长抑制素（MSTN）是一种负调控骨骼肌生长的因子。本研究的目的是在大肠杆菌中表达牦牛（Bos grunniens）重组 MSTN 蛋白，并研究其免疫原性特征。通过反转录 PCR 扩增编码牦牛 MSTN 成熟肽的 cDNA，将其克隆到 pET28a(+)载体中，并在大肠杆菌中表达。通过亲和层析纯化表达的重组 MSTN，并用于制备兔抗牦牛 MSTN 抗体。结果表明，牦牛 MSTN 成熟肽基因包含 330 个核苷酸，编码 109 个氨基酸。当 MSTN-pET28a(+)-BL21(DE3)菌在含有 0.1mM IPTG 的 LB 培养基中孵育 6 小时时，目标蛋白的含量占总表达产物的 21%。纯化的牦牛 MSTN 重组蛋白的分子量为 16.5kDa，ELISA 结果表明其具有优异的免疫原性。所得的牦牛重组 MSTN 适合进一步分析牦牛 MSTN 的功能。