VZV外膜蛋白E胞外区的原核表达及其免疫特性研究

Prokaryotic Expression of VZV Outer Membrane Protein E Extracellular Domain and Study of Immune Characteristics

水痘-带状疱疹病毒(VZV)是一种可以引起水痘和带状疱疹(HZ)的病原体,通过呼吸道和密切接触在人群中传播,可以引发水痘和带状疱疹两种不同临床症状的疾病。人群对该病毒易感性强,主动免疫是减轻带状疱疹疾病负担的重要手段。g E蛋白是VZV抗原性最强、含量最丰富的外膜蛋白,是目前制备带状疱疹亚单位疫苗重要的候选蛋白。该研究通过原核系统表达带状疱疹g E蛋白,筛选合适的佐剂为制备带状疱疹亚单位疫苗提供依据。构建大肠杆菌重组系统表达VZV外膜蛋白E胞外区,用镍离子螯合层析技术纯化目的蛋白,用铝佐剂和Poly I:C佐剂分别与重组蛋白混合后免疫小鼠,检测抗血清免疫效价及相关细胞因子含量来比较佐剂对小鼠免疫应答的影响。构建的大肠杆菌表达系统成功表达目的蛋白,其表达形式主要为包涵体,通过变性复性获得相对分子质量约为63 ku的目的蛋白。免疫后的小鼠产生高效价及高特异性的血清抗体,经检测铝佐剂和Poly I:C佐剂均有较强的的免疫增强效果,并有明显的剂量效应关系。铝佐剂对Th2型细胞因子(IL-5,IL-10)具有更强烈的刺激作用。而对于Th1型细胞因子IL-2的分泌促进,Poly I:C佐剂优于铝佐剂。应用该蛋白抗原加佐剂可以增强机体体液和细胞免疫,该研究为筛选带状疱疹病毒疫苗佐剂及研制亚单位疫苗奠定了基础。该研究为带状疱疹病毒g E蛋白的纯化及佐剂的筛选提供参考,为亚单位疫苗的制备奠定了基础。

Varicella-zoster virus(VZV)is a pathogen that can cause chickenpox and herpes zoster(HZ).It spreads in the population through the respiratory tract and close contact,and can cause two different clinical symptoms of chickenpox and herpes zoster.The population is highly susceptible to the virus,and active immunization is an important means to reduce the burden of herpes zoster disease.g E protein is the outer membrane protein with the strongest antigen content and the most abundant content of VZV,and is an important candidate protein for the preparation of herpes zoster subunit vaccine.This study used the prokaryotic system to express herpes zoster g E protein and screened for suitable adjuvants to provide a basis for the preparation of herpes zoster subunit vaccine.Construct a recombinant E.coli system to express the extracellular domain of VZV outer membrane protein E,purify the target protein by nickel ion chelation chromatography,immunize mice with the recombinant protein using aluminum adjuvant and Poly I:C adjuvant respectively Serum immune titers and related cytokine levels were used to compare the effects of adjuvants on the immune response of mice.The constructed E.coli expression system successfully expressed the target protein,and its expression form was mainly inclusion bodies.The target protein with a relative molecular mass of about 63 ku was obtained by denaturation and renaturation.The immunized mice produced high-titer and highly specific serum antibodies.After testing,aluminum adjuvant and Poly I:C adjuvant have strong immune enhancement effects,and there is a clear dose-effect relationship.Aluminum adjuvant has a stronger stimulation effect on Th2 cytokines(IL-5,IL-10).For the secretion of Th1 cytokine IL-2,Poly I:C adjuvant is superior to aluminum adjuvant.The application of the protein antigen plus adjuvant can enhance the body’s humoral and cellular immunity.This study has laid the foundation for screening herpes zoster virus vaccine adjuvants and developing subunit vaccines.This study provides a reference for the purification of herpes zoster virus g E protein and the selection of adjuvants,laying a foundation for the preparation of subunit vaccines.