优化重叠PCR法进行单链抗体基因扩增和点突变

利用重叠PCR技术扩增单链抗体基因或位点突变是抗体文库构建或稳定表达的关键和难点,国内外文献未见其方法学的系统报道.以不同VH、VL和Linker基因为拼接模板进行重叠PCR,针对影响重叠PCR扩增的拼接类型,引物设计,反应条件等进行优化.结果表明两段重叠连接比三段更容易实现,且扩增效果好;引物的互补序列长度一般应大于15 bp,且在18～24 bp 时扩增效果最好;退火温度在52～60℃,Mg2+浓度在1.5～2.5 mM时对拼接的效果影响较小;直接或间接使用拼接模板均可以实现重叠PCR的扩增.利用优化策略,首次构建了抗除虫菊酯的scFv基因文库并引入抗XAC糖蛋白scFv基因的点突变,为除虫菊酯抗体文库构建和抗XAC重组抗体的稳定表达奠定了基础.

Study of Single Chain Fvs Splicing and Site-directed Mutagenesis Based on Optimal Overlap PCR

Single chain Fvs splicing and site-directed mutagenesis by overlap PCR is a key point in construction of antibody library and stable expression of antibody. Several aspects affecting overlap PCR amplification, including splicing types,primer design,reaction conditions and use patterns, were explored. The results indicated that splicing by two fragments was easier and superior than that of by three fragments usually. Secondly, the length of complementary sequences of primers could be above 15 bp, and 15 to 24 bp was the best in the experiment. Then, annealing temperature from 52℃ to 60℃ and Mg2+ at 1.5～2.5 mM had little influence on splicing reaction and the template used by directly or indirectly was successful for overlap PCR. According to this strategy, we amplified the specific scFv repertories against pyrethrins and also introduced the site-directed mutagenesis of anti-XAC scFv gene successfully, which laid the basis for the construction of anti-pyrethrins scFv library and the stable expression of anti-XAC scFv antibody.