attB位点核心区定点突变对φC31整合酶效率的影响

Effect of Mutations in attB Core Area on the Efficiency of φC31 Integrase

目的考察attB位点核心区碱基突变对φC31整合酶存真核细胞中催化目的基网整合效率的影响。方法选取attB位点核心区中可能对中c31整合酶效率产生影响的碱基，设计含有相应位点突变的引物，采用大引物法两轮PCR扩增得到含突变碱基的attB片段，将其连人含报告基因的载体pEGFP。NI中得到pEGFP—NI—attB。以含野生型attB位点的报告基因载体pEGFP-N1-attB作为对照．将上述载体分别与中φC31整合酶表达质粒共转染HeLa细胞，经过药物筛选、染色、细胞克隆计数后，计算并比较整合效率。结果成功完成9个含有突变碱基attB位点及报告基因载体的构建。酶切及测序鉴定结果显示每个质粒含1或2个突变碱基。其中6个质粒为目标碱基突变，3个为PCR过程中随机产生的非预期突变。细胞实验结果表明，3个含有突变attB报告质粒的整合率与野生型attB相比无显著性差异，其余6个突变attB报告质粒的整合率与野生型attB相比显著降低（P〈0．05），尤其是其中两个attB位点中含有2个碱基突变的报告质粒，整合率降低极为显著（P〈0．001）。结论attB位点核心区碱基保守性较强，其中一些关键位点的突变使得整合酶效率降低。

Objective To investigate the effect of base mutations in attB site core area on the efficiency of φC31 integrase in eukaryotes. Methods Some bases in attB site core area, which may influence the efficiency of qbC31 integrase, were chosen as target bases for mutation. The prim- ers that contain mutations of target bases were designed, and used in two rounds of PCR amplification to obtain attB segments, which carried the mutated bases. Then, the attB segments were linked to pEGFP-N1 to form pEGFP-NI-attB＂, pEGFP-NI-attB, which carries a wild attB site, served as control. The aforementioned plasmids along with the plasmid expressingqbC31 integrase were transfected into HeLa cells. After G418 selection, cell dyeing and colony counting, the integration rate was compared among groups. Results Nine plasmids containing one or two mutations in attB site core area were constructed and identified by restriction enzyme digestion and sequencing. Among them, six plasmids contained the target base mutations, while the other three contained unexpected mutations due to the PCR amplification. Cell experiment showed that the integration rate of the plas- mids that carried the three mutations had no obvious difference from that of the wild type attB-con- rained plasmid, while the plasmids that harbored the other six mutations had significantly lower inte- gration rate than the wild type attB-contained plasmid （ P 〈 0. 05 ） Two attB sites each with two mutations had an extremely lower integration rate than the control plasmid （P 〈 0. 001 ） . Conclu- sion attB site core area is a conserved area for integration mediated by φC31 integrase, and muta- tions of some bases in attB site can decrease the integration rate ofqbc31 integrase significantly.