摘要
以ｐｈａｇｅｍｉｄ，ｐＢｌｕｅｓｃｒｉｐｔ为载体，把牛凝乳酶原基因的ＫｐｎＩ－ＫｐｎＩ片段插入，将牛凝乳酶４５位Ｃｙｓ用Ａｌａ取代，根据遗传密码子的兼并性，在其附近引入限制性内切酶ＣｌａＩ位点．利用寡聚核苷酸介导的定位突变技术，以获得凝乳酶Ｃｙｓ４５Ａｌａ的突变基因．为了提高突变效率，采用Ｋｕｎｋｅｌ等创立的含尿嘧啶单链ＤＮＡ模板法，经酶切鉴定及双脱氧末端终止法测序，证明已获得预期的突变。
The Kpn I-Kpn I fragment of prochymosin gene was inserted into the phagemidvector:pBluescript. Oligonucleotide-directed site specific mutagenesis method was used to sub-stitute the codon of Cys45 of chymosin with Ala. Based on the codon degeneration,a restrictioncleavage site Cla I was introduced alongside the mutation site simultaneously. In order to im-prove the mutation efficiency.the procedure of selection against uracil-containing templatestrand established by Kunkel et al. was used. Restriction site mapping and Taq Track se-quencing (based on dideoxy chain termination and PCR amplification)indicated that the expect-ed mutation was realized.[
出处
《北京联合大学学报》
CAS
1994年第1期33-39,共7页
Journal of Beijing Union University
关键词
凝乳酶原
凝乳酶
定位突变
二硫键
prochymosin,chymosin,oligonucleotide-directed site specific mutagenesis.
