摘要： 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BamHⅠ酶切的猪IL-2基因非定向克隆到BamHⅠ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。
关键词:
猪白细胞介素-2,
克隆,
重组禽痘病毒,
载体,
构建
Abstract: 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 BamHⅠand cloned into pSY538 vector which was digested by BamHⅠand 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 NotⅠ,the pIL-2 fragment containing LP2 and EP2 was achieved and subcloned into pSY681 vecter containing LacZ gene,then the recombinant plasmid pSY681/pIL-2 was developed.The results indicated that the cloned pIL-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.
Key words:
Porcine IL-2,
Clone,
Recombinant fowlpox virus,
Vector,
Construction
中图分类号:
王淑娟, 陈红英, 魏战勇, 刘金朋, 耿静微, 崔保安. 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[J]. 华北农学报, 2011, 26(5): 71-75. doi: 10.7668/hbnxb.2011.05.015.
WANG Shu-juan, CHEN Hong-ying, WEI Zhan-yong, LIU Jin-peng, GENG Jing-wei, CUI Bao-an. Cloning of Porcine IL-2 Gene and Its Construction of Recombinant Fowlpox Viruses Vector[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(5): 71-75. doi: 10.7668/hbnxb.2011.05.015.