半滑舌鳎生长因子midkine的定点改造及原核表达

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

为进一步研究半滑舌鳎midkine(MK)成熟肽活性与功能,对半滑舌鳎生长因子MK成熟肽进行定点改造及原核表达。根据大肠杆菌偏爱密码子的特点,实验利用SOEing PCR法对半滑舌鳎MK成熟肽进行定点突变。将改造后的半滑舌鳎成熟肽MK克隆到载体质粒pET32a(+),构建原核表达载体pET32a(+)-MK,并转化至大肠杆菌BL21(DE3)pLysS菌株中。SDS-PAGE结果显示,与对照组相比,经IPTG诱导表达后半滑舌鳎重组蛋白MK存在于大肠杆菌上清液中,产物相对分子质量约为33 ku。研究表明,本实验对半滑舌鳎生长因子midkine成功进行了定点改造,原核表达质粒成功构建并在大肠杆菌中高效表达,且主要以可溶形式存在。

Midkine is a kind of heparin-binding cytokine, and it promotes growth, survival, migration and other activities of target cells. The expression of fusion protein MK will facilitate further studies for its biological functions in half-smooth tongue sole（ Cynoglossus semilaevis）. Based on the plasmid containing full length of half-smooth tongue sole MK cDNA sequence, the site-directed mutagenesis of predicted mature MK sequence was successfully obtained by SOEing PCR method according to the E. coli codon bias. After being confirmed by sequencing, the resulting gene was cloned to the vector pET32a （ ＋ ） to yield an identified recombinant plasmid pET32a（ ＋ ）-MK,which was then transformed into E. coli host strain BL21 （ DE3 ） pLysS. SDS-PAGE analysis revealed that high level of fusion protein MK was induced by 1 mmol/L IPTG at 37 ℃ and was mainly detected in sonicated supernatant. The yielded fusion protein MK was approximately 33 ku which was in accordance with the theoretical molecular weight. This study establishes the basis for further study for its detailed functions in half-smooth tongue sole.