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目的原核表达H7N9流感病毒血凝素(hemagglutinin,HA)球状区,并评价其免疫原性。方法根据H7N9流感病毒HA晶体结构,设计含有2对和3对二硫键的球状区结构域HA1S(62-284 aa)和HA1L(57-289 aa),扩增后连接至pET-21b(+)载体,构建重组表达质粒pET-21b(+)-HA1S和pET-21b(+)-HA1L,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的包涵体经复性和柱层析纯化获得球状区蛋白。纯化的目的蛋白分别以CpG X1、Al(OH)_3和CpG X1+Al(OH)_3为佐剂免疫BALB/c小鼠,检测小鼠血清抗体水平和中和抗体水平,评价其免疫原性。结果重组表达质粒经双酶切和测序证明构建正确。两个目的蛋白能在大肠埃希菌中表达,经稀释复性和柱层析纯化后均为单体,HPLC检测纯度均大于95%。动物试验结果表明,单独使用CpG X1或Al(OH)_3不能增强免疫应答,同时使用CpG X1+Al(OH)_3能将IgG抗体水平提高3倍左右。体外假病毒中和试验结果表明,中和抗体水平均较低。结论在大肠埃希菌中高效表达了H7N9 HA1S和HA1L两个蛋白,纯化后的蛋白为均一稳定的单体,且具有一定的免疫原性,为进一步研发快速生产H7N9流感病毒基因工程疫苗提供了参考。
Objective To prokaryotic express the globular domain of H7N9 influenza virus hemagglutinin (HA) and evaluate its immunogenicity. Methods HA1S (62-284 aa) and HA1L (57-289 aa) containing 2 pairs and 3 pairs of disulfide bonds were designed according to the crystal structure of HA of H7N9 influenza virus. After amplification, they were ligated to pET-21b (+ (+) - HA1S and pET-21b (+) - HA1L were constructed and transformed into Escherichia coli BL21 (DE3) for expression under IPTG. The expressed inclusion bodies were purified by refolding and column chromatography to obtain globular regions. The purified protein was immunized BALB / c mice with CpG X1, Al (OH) 3 and CpG X1 + Al (OH) 3 as adjuvant respectively, and the serum and neutralizing antibody levels of the mice were tested to evaluate their immunogenicity . Results The recombinant plasmid was confirmed by double enzyme digestion and sequencing. The two proteins of interest can be expressed in Escherichia coli. After purification by dilution and column chromatography, the two proteins were all monomeric. The purity of the two proteins was over 95% by HPLC. Animal experiments show that CpG X1 or Al (OH) _3 alone can not enhance the immune response, while the use of CpG X1 + Al (OH) _3 IgG antibody levels can be increased about 3 times. In vitro pseudovirus neutralization test results show that the neutralizing antibody levels were low. Conclusion The two proteins of H7N9 HA1S and HA1L are highly expressed in Escherichia coli. The purified protein is a homogeneous and stable monomer with a certain degree of immunogenicity, which provides a basis for further research and development of a genetically engineered vaccine for rapid production of H7N9 influenza virus reference.