牛凝乳酶原基因在食品级乳酸乳球菌中的重组表达

Recombinant Expression of the Bovine Prochymosin Gene in Food-Grade Lactococcus lactis

将牛凝乳酶原基因连接pNZ8149载体,并转化乳酸乳球菌NZ3900,经乳酸链球菌素Nisin诱导,测得重组菌株胞内凝乳酶活力达到0.7 SU/mL,培养基中检测不到凝乳酶活力,实现了牛凝乳酶原基因在乳酸链球菌Nisin诱导基因表达系统(nisin controlled gene expression system,NICE)中活性表达。在此基础上,将分泌信号肽SPusp45连接于pNZ8149,构建了分泌型表达载体pNZ8149s,并实现牛凝乳酶原基因在NICE系统中分泌表达。当使用1 ng/mL Nisin诱导5 h后,重组菌株胞内检测不到凝乳酶活力,培养基中凝乳酶活力为1.2 SU/mL,说明pNZ8149s能够促使凝乳酶原从乳酸乳球菌中分泌。该方法为重组牛凝乳酶在食品级菌株中重组表达提供了一种可行的方案。

In this study,we cloned the bovine prochymosin gene pcm into the expression vector pNZ8149 and transformed into Lactococcus lactis NZ3900.The intracellular chymosin activity attained 0.7 SU/mL in the presence of nisin induction,suggesting that the bovine chymosin gene was successfully expressed in the food-grade nisin controlled gene expression system （NICE）.Furthermore,the secretion signal SPusp45 was ligated into pNZ8149 to generate pNZ8149s.Recombinant L.lactis containing pNZ8149s-pcm showed a chymosin activity of 1.2 SU/mL in culture supematant after induction by 1 ng/mL nisin for 5 h.This result demonstrated that bovine chymosin could be secreted by the expression vector pNZ8149s.This study provides a feasible method for producing recombinant bovine chymosin in food-grade strains.