黄鳍鲷白细胞介素1基因原核表达载体的构建
The construction of two recombinant expression vectors of interleukin-1 gene from Acanthopagrus latus
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摘要: 根据黄鳍鲷白细胞介素1（interleukin-1,IL-1）基因全长cDNA序列（GenBank登录号为AY669059）设计、合成1对特异性引物，扩增编码黄鳍鲷IL-1基因前体肽的基因序列，通过T-A克隆构建了克隆载体pMD18T-IL1。以克隆载体pMD18T-IL1为模板，以设计合成的带酶切位点的引物分别扩增黄鳍鲷IL-1的前体肽和预测的成熟肽基因序列，经BamHI和SalI双酶切后将其插入表达载体pQE30中，构建了原核表达质粒pQE30-pIL1和pQE30-mIL1。经酶切、PCR鉴定并最终通过序列测定表明，基因已正确插入到载体的多克隆位点，序列和读码框都正确无误，为黄鳍鲷IL-1基因的体外重组表达研究打下了基础。Abstract: A pair of primers were designed and synthesized according to the interleukin-1 (IL-1) gene sequence (GenBank accession number is AY669059) of the yellowfin seabream Acanthopgrus latus (Houttuyn). The plasmid pMD18T-IL1 was constructed after amplifying open reading frame cDNA sequence of the IL-1 gene by RT-PCR. The sequence of propeptide and predicted mature peptide were linked into expression vector pQE30 and two recombinant plasmids pQE30-pIL1 and pQE30-mIL1 were constructed. Restriction analysis, PCR amplification and sequencing demonstrated that the target fragments were inserted into pQE30 correctly.
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