重组4-1BB配体对治疗性重组人乳头瘤病毒16型蛋白疫苗的佐剂作用

摘要

目的 研究重组4-1BB配体(recombinant 4-1BB ligand,r4-1BBL)作为佐剂对治疗性重组人乳头瘤病毒16型(human papillomavirus-16,HPV16)蛋白疫苗(HPV16蛋白疫苗)免疫效果的影响.方法 对4-1BBL胞外功能区进行密码子优化,并通过NdeⅠ和XhoⅠ酶切位点定向插入pET28a表达载体.将获得的重组表达载体转化至大肠埃希菌BL21 (DE3),并用异丙基-β-D-硫代半乳糖苷诱导重组蛋白表达.用蛋白质印迹法对重组蛋白进行鉴定,并用非还原性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电子显微镜分析重组蛋白结构.用CCK-8试剂盒检测纯化r4-1BBL对小鼠T细胞体外增殖的影响.同时将r4-1BBL与HPV16蛋白疫苗联合免疫C57BL/6小鼠,用酶联免疫斑点试验检测小鼠的细胞免疫应答水平,观察r4-1BBL对HPV16蛋白疫苗抑制肿瘤生长的影响.结果 密码子优化的4-1BBL胞外功能区能在重组表达载体中表达,且表达产物同时以包涵体和可溶性形式存在.可溶性产物的结构主要为二聚体,电子显微镜下可以观察到类似亚单位的结构.纯化r4-1BBL可促进小鼠脾淋巴细胞的体外增殖.与单纯HPV16蛋白疫苗相比,r4-1BBL联合HPV16蛋白疫苗能诱导更强的特异性细胞免疫应答(t=3.525,P=0.024)和产生更明显的肿瘤生长抑制作用(t=2.534,P=0.021).结论 r4-1BBL能在小鼠中提高HPV16蛋白疫苗的免疫效果,具有作为HPV16蛋白疫苗佐剂的潜力.%Objective To study the adjuvant effect of recombinant 4-1BB ligand (r4-1BBL) on therapeutic recombinant human papillomavirus-16 (HPV16) protein vaccine.Methods The coding sequence of 4-1BBL extracellular functional domain was optimized and cloned into the pET28a vector at NdeⅠ and XhoI sites directionally.The recombinant plasmid pET28a-4-1BBL was transformed into E.coli BL21 (DE3),and protein expression was induced with IPTG.Expression product was identified by western blotting,and protein structure was analyzed by non-reducing SDS-PAGE and electron microscopy.CCK-8 kit was used to detect the effect of r4-1BBL on mouse T cell proliferation in vitro.C57BL/6 mice were immunized with a combination of r4-1BBL and HPV16 protein vaccine,and the level of cellular immune responses in mice were detected by ELISPOT,and the effect of r4-1BBL on tumor growth inhibition by HPV16 protein vaccine was observed.Results The codon optimized 4-1BBL extracellular functional domain could be expressed in recombinant plasmid pET28a-4-1BBL.The expressed proteins existed as inclusion body and soluble form simultaneously.The structure of soluble proteins was mainly dimer and subunit-like structure was observed under electron microscope.The purified r4-1BBL enhanced the proliferation of mice spleen lymphocytes in vitro.In comparison with HPV16 protein vaccine alone,HPV16 protein vaccine combined with r4-1BBL induced stronger T cell immune responses (t =3.525,P =0.024) and higher tumor growth inhibition (t =2.534,P =0.021).Conclusion r4-1BBL can improve the immune effect of HPV16 protein vaccine in mice,and has the potential as an adjuvant for HPV16 protein vaccine.