摘要
根据黄鳍鲷白细胞介素1β(interleukin-1β,IL-1β)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-1β基因前体肽的基因序列,通过T-A克隆构建了克隆载体pMD18T-IL1β。以克隆载体pMD18T-IL1β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-1β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-pIL1β和pQE30-mIL1β。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-1β基因的体外重组表达研究打下了基础。
A pair of primers were designed and synthesized according to the interleukin-1β (IL-1β) gene sequence (GenBank accession number is AY669059) of the yellowfin seabream Acanthopgrus latus (Houttuyn) . The plasmid pMD18T-IL1β was constructed after amplifying open reading frame cDNA sequence of the IL-1β gene by RT-PCR. The sequence of pro-peptide and predicted mature peptide were linked into expression vector pQE30 and two recombinant plasmids pQE30-pIL1β and pQE30-mIL1β were constructed. Restriction analysis, PCR amplification and sequencing demonstrated that the target fragments were inserted into pQE30 correctly.
出处
《南方水产》
2005年第3期37-41,共5页
South China Fisheries Science
基金
广东省"十五"攻关项目(99B06103G)
关键词
黄鳍鲷
IL-1Β
表达载体
构建
Acanthopgrns latus
interleukin-1β
expression vector
construction