The construction of two recombinant expression vectors of interleukin-1β gene from Acanthopagrus latus
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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。
Abstract: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.
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Keywords:
- Acanthopgrus latus /
- interleukin-1β /
- expression vector /
- construction
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图 1 表达载体pQE30-pIL1β和pQE30-mIL1β的构建示意图
Figure 1. Construction of recombinant expression plasmid pQE30-pIL1β and pQE30-mIL1β
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图 2 重组质粒pMD18T-IL1β的酶切鉴定
M.100 bp DNA分子量标准; 1.BamHI+SalI消化后的重组质粒
Figure 2. Identification of recombinant plasmid pMD18T-IL1β
M.100 bp DNA marker; 1.Digested recombinant plasmid pMD18T-IL1β by BamHI and SalI
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图 3 重组质粒pQE30-pIL1β (a)和pQE30-mIL1β (b)的酶切鉴定
M.100bp DNA分子量标准, 1. BamHI+SalI消化后的重组质粒；2. BamHI+SalI消化后的载体pQE30
Figure 3. Identification of recombinant plasmid pQE30-pIL1β and pQE30-mIL1β
M. 100 bp DNA marker; 1.Digested recombinant plasmids by BamHI SalI; 2.Digested pQE30 by BamHI and SalI
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图 4 黄鳍鲷IL-1β氨基酸序列与其它鱼类IL-1β氨基酸序列CLUSTAL比对分析结果
箭头所指为推测的成熟蛋白切点，相同和相似氨基酸残基分别用“*”和“.”或“: ”表示
Figure 4. Alignment of the predicated yellowfin seabream IL-1β translation with other known IL-1βs
Identical (*)and similar (: or.)residues identified by CLUSTAL are indicated, predicated mature protein cut site is indicated with an arrow.
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