牛凝乳酶原基因在乳酸乳球菌中的表达

【目的】利用乳酸乳球菌nisin诱导基因表达系统(the NIsin Controlled gene Expression system,NICE)表达牛凝乳酶原。【方法】从克隆载体pS19-PPC中获得牛凝乳酶原基因,将该基因与表达载体pNZ8148连接并电转化乳酸乳球菌NZ9000,转化子经酶切、PCR和测序鉴定后,用nisin进行诱导表达,表达产物利用SDS-PAGE和Western blot鉴定,表达产物纯化后检测凝乳活性。【结果】重组牛凝乳酶原与天然牛凝乳酶原比较,其分子量大小、免疫性质、生物活性和抑制剂敏感性没有发现显著差异,其凝乳活性可达2×103IMCU/mL。【结论】在乳酸乳球菌中表达了具有凝乳活性的牛凝乳酶原,同时乳酸乳球菌作为发酵剂和凝乳酶产生菌双重角色的实现,为奶酪加工提供了新思路和新方法。

Production of bovine prochymosin in Lactococcus lactis

Abstract:Objective]Bovine prochymosin is expressed by the nisin controlled gene expression system in Lactococcus lactis.Methods]we amplified bovine prochymosin gene from pS19-PPC vector by PCR,and ligated the gene with expression vector pNZ8148.Ligated products were electrotransformated into Lactococcus lactis NZ9000.Then we identified transformants by restriction,PCR and sequencing, and molecular weight and immune characteristics of expression product by SDS-PAGE and Western blot after inducting using nisin.Eventually we tested milk-clotting activity after purification.Results]Recombinant bovine prochymosin was not significant difference in molecular weight,immune characteristics, bioactivity and sensitivity of inhibitors comparison to nature bovine prochymosin.The milk-clotting activity of recombinant bovine prochymosin was 2×103 international milk-clotting unit per millilitre.Conclusion]We expressed recombinant bovine prochymosin of milk-clotting activity in Lactococcus lactis. This study suggested potential application in cheese manufacture as a new method,which could regard Lactococcus lactis as starter of milk and host of expression bovine prochymosin.