炭疽毒素受体ATRcDNA的合成和克隆及ATR-Fc融合蛋白的真核表达载体的构建

Chemical Synthesis and Cloning of cDNA of Anthrax Toxin Receptor (ATR)and Construction of Eukaryotic Expression Vector for ATR-Fc Fusion Protein

可溶性炭疽毒素受体(sATR)可以特异性结合炭疽毒素保护抗原(PA),为获得用于中和炭疽毒素以防治炭疽感染的候选抗毒素药物,构建了表达ATRFc抗体样分子融合蛋白的真核表达载体。将全长为681bp的编码炭疽毒素受体N端1～227氨基酸的基因分成长约50～60碱基的18个寡核苷酸片段,相邻片段重叠部分为20～22个碱基,利用重叠延伸PCR和引物PCR法,将合成的片段组装与扩增,得到了含有ATR1～227的全部编码区和HindIII、BamHI位点在内的DNA片段。回收的基因片段经BamHI/HindIII双酶切连接到pUC19质粒中,挑选阳性克隆进行酶切鉴定和双向序列测定,获得了全序列正确的克隆。将ATR基因与Fc基因连接后插入pcDNA3.1载体多克隆位点HindIII和NotI之间,得到表达ATRFc融合蛋白的真核表达载体pcDNA3.1/ATRFc,为利用CHO哺乳动物细胞表达ATRFc并研究其生物学性质奠定了基础。

Soluble anthrax toxin receptor （sATR） can bind specially and directly to protective antigen （PA）, a component of anthrax toxins which plays a central role in the toxin-mediated pathogenesis of anthrax infection. In order to find a candidate for the antitoxin that can neutralize and block PA, a eukaryotic vector for expression of ATR-Fc, an antibody-like fusion protein, was constructed. The cDNA, encoding N-terminal amino acids 1 - 227 of anthrax toxin receptor （ATR） and consisting of 681base-pairs, was divided into 18 oligonucleotide fragments with about 50 - 60 bases length and 20 - 22 bases crossover. The synthetic 18 fragments were assembled and amplified by overlap extended PCR and primer PCR, and the PCR product was cloned into the BamH I and Hind III sites of pUC19 to generate pUC19/ATR. Positive clones were selected, and the whole synthetic gene of ATR was correct, which was confirmed by DNA sequencing. The DNA fragment encoding ATR-Fc was inserted into the Hind III and Not I sites of pcDNA3.1 （ ＋ ） to generate the eukaryotic vector pcDNA3. 1/ATR-Fc, and was the foundation for expression of ATR-Fc fusion protein and further investigation of the biological characterization of ATR-Fc.