【作者】 肖海燕；

【导师】 王向阳； 顾青；

【作者基本信息】 浙江工商大学 ， 农产品加工与贮藏， 2008， 硕士

【摘要】 黑芥子酶（EC3.2.1.147）存在于十字花科植物中。在一定的条件下,黑芥子酶水解硫代葡萄糖苷,生成葡萄糖、硫酸盐、异硫代氰酸盐、乙腈、硫氰酸盐等物质。其中,酶解产物异硫代氰酸盐具有很好的抗肿瘤生物活性。但异硫代氰酸盐在水相中不稳定,易降解失去生物活性。硫代葡萄糖苷的稳定性较好,且黑芥子酶被固定化后,也有很好的稳定性。不过从植物中提取黑芥子酶,产量低,不能满足产品生产的需要。因此,我们利用转基因技术构建合适的工程菌,以期得到大量的、纯度高、有生物活性的黑芥子酶。本实验中,我们尝试将油菜中MYR1基因克隆到原核表达载体中进行异源表达。为了高效表达黑芥子酶,我们采用了pGEX-4T-1表达系统。实验首先从油菜幼苗中提取总RNA,采用RT-PCR法将MYR1编码的成熟肽链基因序列扩增出来。经过克隆测序,与NCBI序列库对比同源性,得知该基因与NO.60214有99%的同源性,氨基酸仅有一个不同。再利用SalⅠ位点与pGEX-4T-1连接,构建成重组子pGEX-4T-1-MYR1。经过PCR鉴定、SalⅠ酶切及EcoRⅠ酶切鉴定以及测序,筛选出重组大肠杆菌。将之在在OD600为0.8、37℃、IPTG浓度为0.08mmol/L的条件下诱导表达2.5h,表达产物经SDS-PAGE初步检测,与空质粒pGEX-4T-1表达产物相比较,其在90KDa处有表达量很高的一条带,但没有获得可溶性的GST融合蛋白,推测是GST融合蛋白大量表达聚集形成了包涵体。通过改变培养条件,在20℃、OD₆₀₀为0.8左右、IPTG浓度0.02mM下诱导2.5h,液氮破碎后经过浓缩,再用少量的pH6的磷酸缓冲液溶解,我们获得少量可溶的、有活性的GST融合蛋白,以SINIGRIN做底物,测得融合蛋白的比活力为3.1×10^-3U/mg。更多还原

【Abstract】 Myrosinase（EC3.2.1.147）,which exists in Cruciferae family,can hydrolyze glucosinolates into glucose、sulfat、isothiocyanates、nitrile and thiocyanate.Thereinto,The enzymatic products of isothiocynate have several biological activities.But the isothiocynate is unstale in water,it easily degrade and lose activities.Glucosinolates have a good stabilization,and also myrosinase have a good stabilization by immobilized.But the economic efficiency of withdrawing myrosinase from the plant is low,cannot satisfy the production needed.Therefore,we use transgene technology to construct the appropriate project bacteria,to obtain high purity and activity myrosinase.In this experiment,we attempt to hetorologous expression throug the MYR1 gene cloning to prokaryotic expression vector.In order to highly effective expresses myrosinase,we use the pGEX-4T-1 express system.At first,we withdraw total RNA from the rape seedling,uses the RT-PCR to expand the MYR1 encoded mature peptide gene sequence increases.By sequencing after the clone,contrasting with the NCBI sequence,we know this gene there has 99%homology with NO.60214, the amino acid only one is difference.Again,we use the SalⅠspot to connect with the pGEX-4T-1,constructing recombinant pGEX-4T-1-MYR1.Confirmating by PCR,SalⅠenzyme cutter and EcoRⅠenzyme cutter、sequencing,We screen the correct recombinant.Expression product at OD₆₀₀0.8、37℃、0.08mmol/LIPTG、and 2.5h,and it expressed highly at 90KDa contrasting with the product of pGEX-4T-1 when run in the SDS-PAGE.But it had not obtained the soluble GST fusion protein,by supposing the GST fusion protein massive expressions gathering and forming inclusion body.After changing conditions,we induced 2.5h at 20℃,OD₆₀₀0.8,IPTG 0.02mM,after lysised cell by fluid nitrogen and concentrated,then used a little of phosphoric buffer（pH=6）.Finally,we obtain a little of dissolve activity GST fusion protein,as SINIGRIN is substrate,The specific activity of the fusion protein is 3.1×10^-3U/mg.更多还原