摘要
以实验室保存的携带凝乳酶原前体基因的重组载体pMD 19-T/bPPC为模板克隆凝乳酶原基因,经双酶切后与载体pET-30a连接得到重组载体pET-30a/bPC,转化大肠杆菌BL21(DE3),经IPTG诱导后,采用SDS-PAGE检测目的蛋白表达情况。重组蛋白经变性/复性、DEAE-Sepharose Fast Flow纯化和自催化后检测凝乳活性。结果表明,重组凝乳酶原基因在大肠杆菌中高效表达,表达量占菌体总蛋白的68%,采用Arima K方法检测,其凝乳活力达到80 SU/mL。因此,通过大肠杆菌表达系统大量制备具有生物活性的重组牛凝乳酶原的策略是可行的,研究结果为弥补国内天然牛凝乳酶的短缺提供一种途径。
The bovine prochymosin gene was cloned from the recombinant vector of pMD ~ 19-T/bPPC harboring the interest gene, and which was then ligated with the expression vector pET-30a. Ligated products were transformed into E. cotl BL21 （DE3）. Recombinant protein was analysed by SDS-PAGE after the IPTG induction. The recombinant bovine chymosin was then used to assay the milk-clotting activity after denaturation/renaturation, DEAE-Sepharose Fast Flow purification and activation. The recombinant prochymosin was high-efficiently expressed in E. coli, making up 68% of the total protein. The milk-clotting activity of recombinant chymosin was 80 SU/mL using the Arima K method. So it is possible to largely produce bioactive recombinant bovine prochymosin through the E. coli expression system. These results would provide a method to solve the problem of lack of authentic bovine chymosin.
出处
《中国乳品工业》
CAS
北大核心
2012年第3期4-6,共3页
China Dairy Industry
