犬γ干扰素基因的克隆与序列分析

Cloning and Identification of Canine Interferon Gene

根据已发表的犬γ干扰素 (Ca-IFN -r)cDNA序列设计引物 ,用植物血凝素 (PHA)有机锗 (Ge - 1 3 2 )刺激体外培养的犬脾细胞产生IFN ,提取IFNmRNA ,并以其为模板进行返转录聚合酶链反应 (RT -PCR) ,扩增出 4 80bp基因片段。经酶切鉴定后将其克隆到pUC1 9的SmaI,构建重组质粒 pUC·IFN。经酶切、PCR及序列分析鉴定 ,可证明克隆的基因为犬γ干扰素 ,与所发表的核苷酸序列同源率为 97.3 %

A pair of primers was designed according to the gene sequence of canine IFN-γ and synthesized.Canine spleen cells were induced with PHA and Ge-132 for 12～24h in vitro,and mRNA of IFN-γ were extracted.Reverse transcription polymerase chain reaction(RTPCR)was performed to amplify 480 bp fragment of the IFN-γ gene.The PCR product was cloned into SmaI site of pUC19 plasmid and the recombinant plasmid vector(pUC-IFN)was abtained.PCR was used to identify the positive recombinant pUC-IFN and 480 bp DNA fragment was amplified.The digestion results confirmed that the cloned gene was identical with the previously published Canine IFN-γ gene,rate of homology is 97.3% with enunciable sequence.This study is a foundation for further research on the molecular biology of Canine IFN-γ and for construction of adjuvant vaccine by means of genetic engineering.