人呼吸道合胞病毒F蛋白在大肠杆菌中的表达与免疫效果研究

Expression of Human Respiratory Syncytial Virus F Protein in Escherichia coli and Its Use as a Vaccine

人呼吸道合胞病毒(Human respiratory syncytial virus,hRSV)是婴幼儿和老年人发生严重呼吸道疾病的主要病因,自上世纪50年代hRSV被发现以来,科学家对hRSV疫苗进行了大量的实验探索,但至今没有上市的疫苗。本研究应用pET32a质粒在大肠杆菌BL21中表达并纯化了hRSV F蛋白(RBF),透射电镜下RBF以三聚体的形式存在(长约20 nm的棒状结构),与5C4抗体(识别抗原表位Ø)亲和常数为1.26×10−9。混合铝佐剂肌肉注射免疫BALB/c小鼠,不同剂量(2.5μg、5μg和10μg)的RBF蛋白免疫效果无显著差异(P>0.05),均能诱导小鼠产生高滴度的中和抗体(滴度可达27),并且可以显著降低肺脏病毒滴度,减少肺脏的病理损伤(P<0.001)。与福尔马林灭活疫苗(FI-RSV)相比,RBF诱导产生偏向Th1类的细胞和体液免疫应答。RBF在动物实验中显示出良好的保护效果,同时原核表达系统具有产量和成本上的优势,因此可以作为预防人感染hRSV的潜在候选疫苗。

Human respiratory syncytial virus(hRSV)is a primary cause of severe respiratory tract disease in infants and the elderly.Since the discovery of hRSV in the 1950s,scientists have conducted many experiments aiming to produce an hRSV vaccine,but there is no vaccine on the market.In this study,we expressed the hRSV F protein(RBF)in Escherichia coli BL21 and purified it.RBF exists as a trimer with some prefusion F conformation visible under an electron microscope.The affinity constant between RBF and 5C4 antibody(identify antigen epitopeØ)is 1.26×10−7.Intramuscular injection of BALB/c mice with aluminum adjuvant and RBF protein at different doses(2.5μg,5μg,and 10μg)resulted in no significant difference in the immune effect,which could induce mice to produce high-titer neutralizing antibody(titer up to 27)and significantly reduce pulmonary virus titer and pathological damage in the lungs(P<0.001).RBF vaccine induced a Th1-like cellular and humoral immune response.RBF has shown good safety and efficacy in animal studies,with both yield and cost advantages,making it a potential candidate vaccine for the prevention of hRSV infection in humans.