人白介素-2重组基因的T载体和pET28a表达载体的构建

从人单核细胞中提取总RNA，利用RT－PCR方法构建cDNA—mRNA杂交链，然后用人白介素－2基因的特异引物进行PCR，PCR产物回收后与T载体连接，经过转化、质粒酶切、测序等一系列手段对插入情况和序列是否正确进行鉴定．鉴定正确后用另一组带有酶切位点的引物和pfu酶进行PCR，用EcoR Ⅰ和Hind Ⅲ双酶切PCR回收片段及pET28a表达载体，二者经过连接、转化、质粒酶切、测序等手段重新鉴定序列是否正确．结果表明，插入列T载体和pET28a载体中的序列为402 bp的不带信号肽的基因片段，此序列和GenBank中公布的IL-2序列相一致，故从人单核细胞中提取mRNA后，利用RT-PCR构建的人IL-2基因序列完全正确，为下一步的工作提供了基础。

Constructing and Sequencing T Vector and pET28a Expression Vector of Interleukin-2 gene

Abstract： T Vector and pET28a expression vector of recombinant Interleukin-2 （IL-2） were constructed and sequenced for next step work of protein expression and construction of other IL-2 fusion gene. Total RNA was prepared from human placenta, cDNA-mRNA hybrid was constructed by RT-PCR, then PCR with special primers of IL-2, the product of PCR was recycled and ligated to T vector, digested the plasmid DNA of the selected transformantion with an appropriate restriction endonuclease, the plasmid DNA was sequenced to check for mutation which is introduced by PCR. After being determined that the fragment was inserted correctly, PCR again with pfu enzyme and another primers, and the PCR fragment of IL-2 and pET28a vector were digested with two restriction endonuclease of EcoR Ⅰ and Hind Ⅲ, then following the same procedure of ligating, transforming, sequencing to confirm fragment introduced into pET28a-vector. The DNA fragment introduced into the two kinds of vectors is a fragment 402 bp gene which is no signal peptide coding sequence. The sequence of IL-2 by RT-PCR from the mRNA of human single nuclear cell matches well with that in the GenBank, this result laid a sound foundation for the work of next step.