猪IL-18 mRNA定量RT-PCR检测方法的建立

根据GenBank中猪白介素(IL)-18基因序列,设计1对引物,用RT-PCR技术从猪肺泡巨噬细胞总RNA中扩增出其411 bp cDNA片段,将其与pGEM-T Easy载体连接,经转化、筛选阳性克隆、酶切与测序鉴定后,构建出含猪IL-18 cDNA部分片段的重组质粒。后用反向PCR技术构建出与扩增411 bp片段共用同1对引物,但缺失174 bp的竞争重组质粒。通过上述2种质粒的竞争PCR方法,建立了猪IL-18 mRNA的标准竞争曲线,得到其直线回归方程^y=0.583 2x-2.903 4(R2=0.9898)。

Development of a Quantitative RT-PCR Assay for Porcine IL-18 mRNA

A pair of primers for porcine interleukin(IL)-18 gene was designed according to its sequence available in GenBank,and a cDNA segment of 411 base pair(bp)was amplified by RT-PCR method from the total RNA of porcine alveolar macrophages.After the ligation of the 411 bp cDNA segment with pGEM-T Easy vector and transformation,some positive clones was selected and identified via restrictive enzyme and sequence analysis.Then,one recombinant plasmid(primitive plasmid) contained the 411 bp cDNA segment was obtained.The other recombinant plasmid,containing 237 bp of the 411 bp segment,could be amplified by the same primer,was generated by reverse PCR method based on the primitive plasmid as a competitive depletion clone(competitive plasmid).Based on the competitive PCR between the primitive and the competitive plasmid,the standard competitive curve for porcine IL-18 mRNA was developed,and its linear regression equation as y=0.583 2 x-2.903 4(R2=0.9898) was obtained.