摘要
目的:利用DNA改组技术进行不同亚型的HCVC和E1区的人工进化。方法:首先利用PCR扩增两段具有较高序列同源性的1kb左右的基因片段,两者相比较同源性大于83%。然后将其等量混合,在Mg2+存在的条件下,用DNaseⅠ切割成50bp左右的小片段。这些小片段在不外加引物的条件下,利用PCR反应进行重聚,再将重聚物经过一轮正常的PCR扩增。结果:获得了与原来片段大小相当的基因片段。结论:这一技术为进一步筛选高活性的HCVC和E1区打下基础,有利于从一组序列同源性程度较高的基因库构建随机嵌合基因,并为改组其他基因家族提供了借鉴。
Objective：To use DNA shuffling for directed evolution of HCV C and E1 with different genotypes.Methods： Two genes of about 1 kb were obtained by PCR from two different genotype templates, respectively, which shared over 83% homology. After mixed with equimolar of each, these two genes were further cut randomly by DNase I into small fragments of about 50 bp under the existence of Mg^2＋ .Results： These small fragments were successfully reassembled to form full genes with original size by one round of PCR without any external primers and another round of normal PCR amplification. Conclusion： This shuffling protocol has laid the foundation for further experiments in the screening for high activity of C and El. It may also help to construct chimera genes from a family of genes with high sequence homology and be used to evolve other gene families.
出处
《中西医结合肝病杂志》
CAS
2005年第6期343-345,共3页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
基金
国家自然科学基金资助项目(No.30300301)
