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

Adjuvant effect of recombinant 4-1BB ligand on therapeutic recombinant human papillomavirus-16 protein vaccine

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（humanpapillomavirus16，HPVl6）蛋白疫苗（HPVl6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-IBBL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r4-1BBLL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。

[Abstract] Objective To study the adjuvant effect of recombinant 4-1BB ligand （r4-1BBL） on therapeutic recombinant human papillomavirus-16 （HPVI6） 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-lBBL 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, FIPV16 protein vaccine combined with r4-IBBL 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 HPVI6 protein vaccine.