To realize the prokaryotic expression and explore its reasonable expression conditions, the study subcloned the coding DNA for the mature protein of rabbit BMP7,and constructed the Engineered Escherichia Coli pET32a-mBMP7/BL21 ( DE3 )and pET32a-mBMP7/BL21 (DE3) pLysS, respectively. The effects of culture temperature, IPTG levels and induction time on the expression of desired fusion protein were also analyzed. The results showed that the interest fragment of DNA coding the mature protein of rabbit BMP7 was correctly inserted into the prokaryotic expression vector pET32a. under the induction of IPTG, the both engineered Escherichia Coli produced the desired 34 KD fusion protein consisting of the target mature protein as well as a vector tag protein,and the output of fusion protein reached to nearly 30％ of the total protein of Escherichia Coli. The comparative expressions implied that a higher amount of fusion protein could be harvested when the hosts were cultured at the temperature of 37 ℃ ,and with the use of a IPTG level of 0.75 mM as well as with a induction time of 150min,while at a lower temperature (20 ℃ ) culture,the 0.2 mM IPTG and a induction time of 7 h seemed to be suitable. In addition ,the foreign protein was found not to be deleterious to the growth of host. It then been concluded that the engineering Escherichia Coli are successively constructed, and suitable conditions for the expression of target protein are proposed. These will lie a good foundation to the further studies on the either structure or the functions of this proteins.%为实现家兔BMP7基因原核表达及探索适宜的表达条件,采用PCR技术从pMD18-T-BMP7重组质粒中扩增获得BMP7基因成熟肽片段(mBMP7),构建原核表达工程茵pET32a-mBMP7/BL21(DE3)和pET32a-mBMP7/BL21(DE3)pLysS,并比较温度、IPTG浓度、诱导时间等因素对目的蛋白表达的影响.结果表明,mBMP7原核表达栽体构建正确;工程茵pET32a-mBMP7/BL21(DE3)和pET32a-mBMP7/BL21(DE3)pLysS经IPTG诱导均可表达含有家兔BMP7成熟肽的34KDa的融合蛋白,表达产物约占茵体总蛋白的30%.在37℃、0.75 mM IPTG以及诱导150 min时,能取得较高的目的蛋白表达量;在20℃低温条件下,IPTG浓度和诱导表达时间应分别以0.20mM和7 h为宜;另外,目的蛋白表达对工程茵生长无明显的不利影响.因此,成功地构建了家兔BMP7基因工程茵,并确定了适宜的表达条件,为进一步研究该蛋白的结构和功能奠定基础.
展开▼
