猪白细胞介素-2基因的克隆及其重组禽痘病毒载体的构建

摘要

Porcine IL-2 gene was amplified by RT-PCR from the total RNA extracted from the splenocyte of Yu' nan pig. The PCR product was cloned and sequenced. The porcine IL-2 (pIL-2) gene was digested by Bamli I and cloned into pSY538 vector which was digested by Bamli I and dephosphorized by calf alkaline phosphatase. By means of the PCR .restriction enzyme digestion and sequencing, the recombinant plasmid pSY538/pIL-2 was selected. After pSY538/pIL-2 was digested by Not I ,the pIL-2 fragment containing LP2 and EP2 was achieved and sub-cloned into pSY681 vecter containing LacZ gene,then the recombinant plasmid pSY681/pIL-2 was developed. The results indicated that the cloned plL-2 gene had 482 bp, including one ORF(465 bp). The IL-2 gene sequence of Yu'nan pig had 99. 9% nucleotide homology with the 5 pig IL-2 genes published in GenBank. Enzyme digestion and DNA sequencing results confirmed that the sequence of the recombinant plasmid pSY681/pIL-2 contained the target fragment,and the ligation part was correct. The results showed that the recombinant plasmid pSY681/pIL-2 was constructed correctly, paving the way for further study of biological function and further application of porcine IL-2.%采用RT-PCR技术自豫南黑猪脾淋巴细胞中扩增猪IL-2基因(pIL-2),RT-PCR产物进行T-A克隆并测序,获得了猪IL-2基因序列.将BamH Ⅰ酶切的猪IL-2基因非定向克隆到BamH Ⅰ酶切并去磷酸化的pSY538载体上,通过PCR、酶切和测序鉴定,筛选正向插入的重组质粒pSY538/pIL-2.NotⅠ酶切pSY538/pIL-2后,得到含有双启动子LP2和EP2的猪IL-2基因片段,将其亚克隆到含有LacZ基因的pSY681载体上,筛选正向插入的重组禽痘病毒转移质粒pSY681/pIL-2.测序结果表明,克隆的豫南黑猪IL-2基因长482 bp,包含1个完整读码框(465 bp),与GenBank 中其他5条猪IL-2基因核苷酸同源性为99.9％.重组质粒pSY681/pIL-2经酶切、测序鉴定,证实含有目的片段,且连接、构建正确.成功构建了重组禽痘病毒转移质粒pSY681/pIL-2,为猪IL-2生物学功能的进一步研究和开发奠定基础.