摘要
为构建长链CpGODNDNA，将合成的含有CpG的GTCGTT＇核心序列以TT间隔开后重复3次，并在3’端添加polyG结构，而后在其两翼人为添加XbaI的黏性末端（简称为A链）后合成与其互补的互补链（简称为B链），通过退火形成CpGODNDNA单体，以DNA连接酶将CpG单体连接为多聚体后再以A链和B链作为引物，利用SOE—PCR技术对polyCpGODNDNA进行长链扩增，分别将600～700bp片段克隆到pMD-18T载体中，并获得相应的重组子pUC44、pUC26及pUC30。序列分析结果表明：3个重组子与理论上产生的含有10，11，13个CTAGATCGTCGTTTTGTCGTIWGTCGTTGAGGGGGGAT拷贝长链CpGODNDNA标准序列的同源性分别为97．4％、97．9％和97．1％。
To construct long strand DNA containing CpG oligodeoxynucleotides, three times repeats of GTCGTT was synthesized and TT was acted as isolated sequence, and polyG were added as tail of the three times repeats. The cohesive end of Xba I was linked closely to polyG （regarded as strand A）, and the complement strand of as synthesized either （regarded as strand B）. The strand A and strand B were annealed to form CpG ODN DNA, and poly CpG ODN DNAs was constructed by T4 DNA ligase, then long strand of poly CpG ODN DNAs were amplified by SOE - PCR with strand A and strand B as a pair of primers and poly CpG ODN DNAs as substrates. The fragments between 600 and 700bp were cloned to pMD - 18T, and three recombinants of pUC44, pUC26 and pUC30 were obtained, the results of the sequences analysis showed homologies to deduced long strand CpG ODN DNA which contained 10, 11 or 13 copies of CTAGATCGTCGTTTTGTCGTIWGTCGTTGAGGGGGGAT were 97.4%, 97.9% and 97.1%, respectively.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2009年第11期10-12,共3页
Heilongjiang Animal Science And veterinary Medicine
基金
佛山科学技术学院重点课题基金项目
关键词
SOE—PCR
长链CpG
ODN
DNA
重组质粒
Splice overlapping extension PCR （SOE -PCR）
long strand CpG ODN DNA
recombinant plasmids
作者简介
作者简介：司兴奎（1973-），男，副教授，博士．
