基于HPV16E7蛋白CTLs抗原肽的病毒样颗粒治疗性疫苗的构建

Development of a HPV16 E7 CTLs Peptides-based Virus-like Particle Therapeutic Vaccine

目的:构建呈递HPV16 E7 CTLs抗原表位的病毒样颗粒,并初步评价病毒样颗粒作为治疗性疫苗载体的潜能。方法:根据文献选择有效的HPV16 E7 CTLs表位,合成其正负链寡核苷酸序列,并通过退火形成双链DNA片段。将片段克隆于表达乙肝核心抗原的重组质粒p Thio His AHBc Ag,使抗原肽得以呈现于病毒样颗粒。重组菌经IPTG诱导后经SDS-PAGE鉴定目的蛋白表达。菌体破碎后经硫酸铵盐析法和蔗糖密度梯度离心进行纯化,并经高效液相凝胶过滤色谱和电镜鉴定病毒样颗粒的存在。制备的病毒样颗粒免疫接种了TC-1细胞的肿瘤模型小鼠,检测小鼠肿瘤大小。此外,在体外以抗原肽刺激脾细胞,以ELISA检测IFN-γ表达水平。结果:构建的三个重组表达质粒经酶切鉴定及测序分析证实构建正确。表达的重组蛋白大小与预期相符,并形成了病毒样颗粒。免疫小鼠后显示了抑制肿瘤增长的一定作用趋势。此外,抗原肽体外刺激促进了疫苗免疫小鼠脾细胞IFN-γ的表达,显示疫苗引起机体产生E7特异性的细胞免疫应答。结论:HBc Ag VLPs是有潜能的治疗性疫苗载体。

Objective ： To construct virus-like particles （VLPs） presenting CTLs epitopes of HPV16 E7, and assess its potentials to be an effective form of therapeutic vaccine. Methods： Three reported effective HPV16 E7 CTLs epitopes were selected and used to be presented by HBcAg VLPs. The oligonucleotides encoding for the three peptides were synthesized and inserted respectively into the plasmid pThioHisA-HBcAg. The recombinant plasmids were tansformed into DHSot cells, and the expression of the chimeric proteins were induced with IPTG and identified by SDS-PAGE. The proteins were purified with a procedure consists of ammonium sulfate precipitation and sucrose density gradient centrifugation, and the presence of VLPs was detected with HPLC of size-exclusion chromatography and electron microscopy. Mice received TC-1 graft were immunized with the mixed VLPs, and the dynamic changes of tumor size were recorded. In addition, the splenocytes isolated from the experimental mice were stimulated in vitro with the synthetic antigenic peptides and the expression of IFN-~/was measured by ELISA. Results： Three constructed recombinant plasmids were proven to be correct by restriction enzyme digestion and DNA sequencing. The recombinant proteins were expressed efficiently, and presented as VLPs. Tumor growth was suppressed in vaccinated mice. Furthermore, the IFN-~/ expression of splenocytes was promoted by the stimulation of antigenic peptides, indicating that the VLPs caused the E7-specific cellular immune response. Conclusion： Presenting HPV E7 CTLs epitopes with HBcAg VLPs is a potential strategy for developing an effective therapeutic HPV vaccine.