金黄色葡萄球菌核酸酶基因的片段缺失与定点突变

FRAGMENT DELETION AND BASE SUBSTITUTION OF STAPHYLOCOCCAL NUCLEASE GENE R

金黄色葡萄球菌核酸酶R(SNase R)是金黄色葡萄球菌核酸酶(SNase A)的一种类似物,具有与SNaseA相同的酶活性,与SNase A唯一不同之处是在N端多出6个氨基酸残基。为了得到完整的SNase基因并使其在E.Coli中表达,我们利用单链U模板—单引物突变法,将为6个额外氨基酸残基编码的18个氨基酸残基删除,其突变率可达90％。进而,完整的SNase A基因被重组入表达载体pBV221。细菌表达产物的PAGE分析结果指出,SNase A在E.coli中得到高效表达。与此同时,我们利用两个不同的引物在单链U模板上同时介导两种不同类型的突变(片段缺失、碱基取代)其突变率可达83％以上,这为进行多种类型的高效突变提供一个有用的方法。本文也对影响突变率的某些实验因素进行了讨论。

Staphylococcal nuclease R (SNase R) is an analogue of Staphylococcal nuc-ease A (SNase A), which has the same enzymatic activities as SNase A and six extra amino acid residues attached to the amino teminus of SNase A. In order to get an exact SNase A gene for expression, the 18 nucleotides coding for the six extra amino acid residues were deleted by uracil-containing single strand phagemid DNA template (U-ss pTZ19R) and one primer method. The mutagenesis frequency was about 90%. Furthermore, the SNase A was inserted into an expression vector pBV 221, at EcoRI/Sall restriction sites. The new expression plasmid was named pBVS-M2. The polyacrylamide gel electrophor-esis analysis results of the cell lysate from host cell harboring the pBVS-M2 showed that the SNase A gene was highly expressed in E. coli.Two different types of mutagenesis (the fragment deletion and hase substitution) were also performed simultaneously according to the same methods described above using two mutagerfetic primers. DNA sequencing analysis indicates that the mutagenesis frequency for two different types of mutagenesis with two different primers was 83.3%. This provides an useful approach to make more than one type of mutagenesis with high efficiency at the same time. Some experimental factors which affect mutagenesis frequency were also discussed.