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 papiilomavirus type 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酶切位点将其插入pET28a表达载体。将获得的重组表达载体转化大肠埃希菌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 NdeⅠand XhoⅠsites 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-reductive 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 were detected by ELISPOT in mice, and the effect of r4-1BBL on tumor growth inhibition of 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 have the potential as an adjuvant for HPV16 protein vaccine.