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构建一种新型抗肿瘤融合蛋白疫苗,包含hVEGF121和βhCG抗原表位,并探索融合蛋白的纯化条件和方法。采用PCR方法,克隆VEGF和βhCG基因,并通过限制性内切酶NcoⅠ、EcoRⅠ和HindⅢ将两个基因逐个插入pET-28a(+)质粒中,利用质粒自带的卡那霉素抗性筛选出阳性克隆,获得重组基因的表达载体。对重组菌进行乳糖诱导表达后,通过超声破碎,硫酸铵分级沉淀,阴离子交换层析等方法进行融合蛋白的分离纯化,Western-blot鉴定。测序结果表明成功构建重组载体pET-28a-VEGF-M2-X10-βhCG-CTP37,重组基因长993 bp,编码331个氨基酸残基。SDS-PAGE显示,经乳糖诱导,Escherichia coli BL21(DE3)重组菌表达hVEGF121-M2-X10-βhCG-CTP37多肽,约占菌体总蛋白量的35%。融合蛋白经分离纯化后纯度可达到电泳纯,约占蛋白冻干粉的80%。结论:克隆、测定了融合蛋白基因,并在Escherichia coli BL21(DE3)中诱导表达hVEGF121-M2-X10-βhCG多肽,同时成功分离纯化此多肽。
To construct a novel anti-tumor fusion protein vaccine containing hVEGF121 and βhCG epitopes and explore the purification conditions and methods of the fusion protein. The VEGF and βhCG genes were cloned by PCR and the two genes were inserted into pET-28a (+) plasmids one by one by restriction endonucleases NcoⅠ, EcoRI and HindⅢ, and then selected by using kanamycin resistance Positive clones to obtain the recombinant gene expression vector. After the recombinant strain was induced by lactose, the fusion protein was isolated and purified by ultrasonic disintegration, ammonium sulfate fractionation and anion exchange chromatography, and identified by Western-blot. The sequencing results showed that the recombinant vector pET-28a-VEGF-M2-X10-βhCG-CTP37 was successfully constructed. The recombinant gene was 993 bp in length and encoded 331 amino acid residues. The result of SDS-PAGE showed that Escherichia coli BL21 (DE3) recombinant expressed hVEGF121-M2-X10-βhCG-CTP37 polypeptide, accounting for about 35% of the total bacterial protein. Purified fusion protein purity can be achieved after electrophoresis pure, about 80% of lyophilized protein powder. CONCLUSION: The fusion protein gene was cloned and the hVEGF121-M2-X10-βhCG polypeptide was induced in Escherichia coli BL21 (DE3). The polypeptide was successfully isolated and purified.