Asia1型FMDV复合表位免疫原的研制及免疫效果研究
摘要
口蹄疫（Foot-and-mouth Disease，FMD）作为一种全球范围内的重大动物疫病，已成为当前动物及其产品国际贸易的主要障碍。O、A、Asia1型FMD是我国一直以来的防治重点，灭活疫苗在预防和控制FMD的过程中发挥着重要作用。
机体抵抗口蹄疫病毒（Foot-and-mouth Disease Virus，FMDV）感染过程中，依赖于中和抗体的体液免疫反应是最主要的。虽然灭活疫苗免疫能够产生高水平的中和抗体，但高水平的中和抗体并不是总能为机体提供完全的保护，同时CD4+T细胞在诱导机体体液抗体水平方面发挥着重要的作用，提示同时提高机体的体液免疫和细胞免疫对于FMD的预防更有意义。复合表位疫苗减少了疫苗中与免疫无关的成分，同时可对T、B细胞抗原表位进行不同组合，是实现口蹄疫体液免疫应答和细胞免疫应答的理想选择。FMDV主要是依靠接触和空气进行传播，黏膜免疫应答能够在黏膜部位产生大量的分泌性的sIgA，分泌性的sIgA不仅可以在第一时间中和病毒，而且可通过黏膜的方式进一步激活系统免疫反应，进而阻止病毒的侵入。所以有效的诱导黏膜部位产生sIgA是口蹄疫疫苗发展的新方向。
本研究人工设计了复合表位肽，并分别以乙型肝炎病毒核心抗原（HBcAg）和牛IgG2a Fc片段为载体，利用原核表达系统和毕赤酵母表达系统，制备三种复合表位免疫原pET28a-HBc-EB、pET22b-EB-BIg和rGS115-pPICZA-HBc-EB，通过腹腔注射、口服和滴鼻三种途径分别对小鼠进行免疫，从体液免疫、细胞免疫和黏膜免疫水平对复合表位疫苗的免疫效果进行综合评价。
（1）Asia1型FMDV复合表位免疫原的设计及合成
选取VP1上第140~160位、第200~211位两个B细胞表位和VP1上第20~40位、3A上第21~35两个T细胞表位为候选表位，并在其间引入合适的linker序列，构成了Asia1型FMDV的复合表位。该复合表位肽通过linker序列GSGSG融合于HBcAg的第78~79位氨基酸（刺突位置），软件分析表明，Linker序列在表位之间形成柔性区域和转角，特别是序列（G4S）3的引入保证了两个B细胞表位的独立作用，而复合表位肽所在的HBc刺突位置具有较高的抗原性且大部分存在于表位部分，并有出现在分子表面的可能性，将融合有复合表位的HBcAg序列优化后合成。
（2）复合表位免疫原在原核表达系统中的制备及腹腔免疫效果评价
1)复合表位免疫原在原核表达系统中的制备
以合成的质粒pUC57-simple-HBc-EB为模板，PCR扩增出目的基因，产物酶切后与pET28a连接，得到重组表达质粒pET28a-HBc-EB。以合成的含有牛IgG2a Fc片段基因的pUC57-simple-BIg质粒为模板，扩增出牛IgG2a Fc片段基因，并利用融合PCR方法与复合表位基因连接，酶切后与pET22b连接，得到重组表达质粒pET22b-EB-BIg。复性后的重组pET28a-HBc-EB在电镜下可观察到病毒样粒子的存在，而重组蛋白pET22b-EB-BIg也形成了类似免疫球蛋白单体的形式，能够与兔抗牛抗体直接结合。Western blot鉴定结果表明两免疫原均具备良好的抗原性，更为重要的是Dot ELISA结果显示复性后的复合表位免疫原能够与Asia1型FMDV VP1构象表位抗体很好的结合，间接说明复合后的表位免疫原能够一定程度上折叠为类似于Asia1型VP1蛋白构象型表位的抗原结构。
2)复合表位免疫原腹腔免疫效果评价
免疫后28d的检测结果表明，pET28a-HBc-EB组和pET22b-EB-BIg组小鼠抗体水平显著高于疫苗免疫组（P＜0.01或P＜0.05），pET28a-HBc-EB组的抗体水平显著高于pET22b-EB-BIg组（P＜0.05）；pET28a-HBc-EB组液相阻断抗体效价高于疫苗免疫组，为1:256~1:512；而pET22b-EB-BIg组液相阻断抗体效价为1:128~1:256，与灭活疫苗组相当；pET28a-HBc-EB组和pET22b-EB-BIg组的淋巴细胞刺激指数显著高于灭活疫苗免疫组（P＜0.01）。pET28a-HBc-EB组和pET22b-EB-BIg组的CD4+/CD8+比值显著高于灭活疫苗组（P＜0.01，P＜0.05）。pET28a-HBc-EB组和pET22b-EB-BIg组小鼠脾细胞刺激后产生的IL-2和IFN-γ显著高于灭活疫苗组（P＜0.01），pET28a-HBc-EB组IL-4和IL-10产生水平显著高于灭活疫苗组（P＜0.05），而pET22b-EB-BIg组与灭活疫苗组无显著差异。表明Asia1型FMDV多表位免疫原具有良好的免疫效果，既可刺激机体产生高水平的体液免疫，又可以刺激机体发生细胞免疫，其中以pET28a-HBc-EB组免疫效果最佳。
复合表位免疫原腹腔免疫较为理想的效果证实了复合表位免疫原的有效性，为口服免疫和滴鼻免疫提供了保证。
（3）复合表位免疫原在毕赤酵母表达系统中的制备及口服免疫效果评价
1)复合表位免疫原在毕赤酵母表达系统中的制备
本研究鉴于毕赤酵母具备真核细胞表达系统的修饰功能的特点，以期获得具有天然结构的复合表位免疫原。酶切重组质粒pET28a-HBc-EB，获得复合表位多肽基因，与pPICZA胞内表达载体连接，获得重组质粒pPICZA-HBc-EB；以本实验保存的含有Asia1型FMDV全基因的质粒为模板，分别扩增病毒衣壳蛋白编码基因，并分别克隆于毕赤酵母表达载体pPICZA中，然后通过PCR方法在含有各表达盒两端引入酶切位点，各表达盒经酶切后依次将连入pPICZA载体中，最终构建酵母表达质粒pPICZA-vp031；Western blot和Dot ELISA鉴定结果表明表达产物具有良好的抗原性。
2)重组毕赤酵母以5×108CFU剂量口服免疫小鼠，免疫后28d检测结果表明，pPICZA-HBc-EB组和pPICZA-vp031组抗体水平显著低于灭活疫苗免疫组（P＜0.01）；液相阻断抗体效价为1:8~1:16；毕赤酵母免疫组小鼠黏膜冲洗液sIgA和特异性sIgA总量显著高于疫苗组（P＜0.01）；pPICZA-HBc-EB组和pPICZA-vp031组的黏膜特异性sIgA显著高于灭活疫苗组（P＜0.01）；pPICZA-HBc-EB组和pPICZA-vp031组淋巴细胞增殖指数均显著高于灭活疫苗免疫组（P＜0.01），而pPICZA-HBc-EB组显著高于pPICZA-vp031组（P＜0.05）；灭活疫苗组CD4+/CD8+比值显著高于pPICZA-HBc-EB组和pPICZA-vp031组（P＜0.01）；pPICZA-HBc-EB组小鼠脾细胞刺激后产生的IL-2和IFN-γ显著高于灭活疫苗组（P＜0.01），而pPICZA-vp031组则与灭活疫苗组无显著差异（P＞0.05），pPICZA-HBc-EB组和pPICZA-vp031组IL-4和IL-10水平与灭活疫苗组无显著差异（P＞0.05）。说明口服重组酵母菌不仅可刺激机体产生体液免疫和细胞免疫，而且还能显著地诱导机体黏膜免疫。证实了复合表位免疫原通过口服免疫激发黏膜免疫的可行性。
（4）复合表位免疫原滴鼻免疫效果的评价
利用原核系统制备的复合表位疫苗对小鼠进行滴鼻免疫，免疫后28d pET22b-EB-BIg组抗体水平显著高于pET28a-HBc-EB组（P＜0.01），显著低于灭活疫苗免疫组（P＜0.01），且抗体上升趋势较为缓慢；液相阻断抗体效价低于1:8；pET22b-EB-BIg滴鼻免疫组黏膜冲洗液总sIgA水平、黏膜特异性sIgA抗体水平显著高于灭活疫苗组、pET28a-HBc-EB滴鼻免疫组（P＜0.01）；pET22b-EB-BIg滴鼻免疫组的刺激指数显著高于其他各组（P＜0.05）；pET22b-EB-BIg滴鼻免疫组CD4~+/CD8~+比值显著低于灭活疫苗免疫组（P＜0.01）。上述结果说明pET22b-EB-BIg免疫后小鼠上支气管和肺部冲洗液、肠冲洗液和生殖道冲洗液中均有特异性sIgA的分泌，同时机体产生一定的体液免疫和细胞免疫，而pET28a-HBc-EB则无此效应，首次实践了靶向FcRn激发黏膜免疫的可行性，为FMD黏膜疫苗的制备提供一种新的思路。
Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral diseaseof cloven-hoofed animals worldwide. Thus, controlling the disease especially caused by the O, A andAsia1foot and mouth disease virus (FMDV) in China through regular vaccination and slaughter ofinfected and contact animals are very important. Currently, the conventional FMD inactivated vaccinehas played an important role in controlling the disease.
Humoral immune response of neutralizing antibodies plays an important role in the anti-FMDVprocess. Production of high titres of neutralizing antibodies induced by the inactivated vaccine is notalways protective in livestock. Stimulating CD4+T-cell responses is important for the induction ofoptimum antibody responses to inactivated vaccine. Therefore, it is essential to consider the efficacy andpractical use of vaccines in order to induce an effective humoral immune response and cell-mediatedimmunity. The epitope vaccines combined with the T cell and B cell epitopes, which reduced theingredients independent within the vaccines is the ideal choice to achieve high humoral and cellularimmune response. The spread of FMD is mainly based on the close contact and the air. The mucosalimmunity induces the mucous membrane to produce many sIgA, which not only can neutralize the virusat the first time, but also induce system immune response. Therefore, effective in inducing the mucousmembranes sIgA is the new direction of development of the foot-and-mouth disease vaccine.
In this study, a multi-epitope peptide was designed, fused to HBcAg and Bovine IgG2a Fc,individually. The three multi-epitope immunogens pET28a-HBc-EB, pET22b-EB-BIg andrGS115-pPICZA-HBc-EB were expressed in E.coli and Pichia pastoris. The three multi-epitopeimmunogens were used to immune mice by intraperitoneal injection, oral and intranasal, then theimmune effects were evaluated by humoral immunity, cellular immunity and mucosal immunity.
(1) Design and synthesis of Asia1FMDV multi-epitope immunogen
In this study, two B cell epitopes (141-160aa and200-213aa) in VP1protein and the T cell epitopes(21-40aa in VP1and21-35aa in3A protein) were linked by the suitable linker sequence between themand synthesized as the candidate contents of the vaccine (the multi-epitopes designated as the EB). ThisEB sequences were inserted to the78to79amino acids of the HBcAg sequence with the linker GSGSG(spike position), and the multi-epitopes designated as the HBC-EB. Based on the software analysis, thelinker sequence formed the flexible regions and corners between the epitopes, especially the sequence(G4S)3guaranteed the two B cell epitopes exist as the independent role. The HBc-EB had a high antigenicity and most of the multi-epitope expressing in the HBc spike position, which was possible insurface. Then, the HBC-EB with its optimized gene sequences was optimized and synthesized.
(2) Generation of multi-epitope immunogen and effects of immunity by intraperitoneal injection
1) Generation of multi-epitope immunogen in E.coli
The target gene was amplified by the PCR and the recombinant expression plasmidpET28a-HBc-EB was constructed. Based on the synthesized pUC57-simple-BIg plasmid as the template,the BIg gene was amplified and fused by the EB gene to generate the recombinant expression plasmidpET22b-EB-BIg. The recombinant plasmids were expressed in E.coli. Electron microscopy showed thatthe refolded protein pET28a-HBc-EB complex formed as virus-like particles. Western blot and DotELISA results confirmed that the recombinant proteins had great antigenicity, and the recombinantprotein pET22b-EB-BIg reacted directly with Horseradish peroxidase-conjugated rabbit anti-bovine IgGantibody. The immunization strategy was done using the antigens based on the Asia1FMDVmulti-epitope immunogen.
2) Effects of immunity by intraperitoneal injection
The test was taken at28d after the mice immunized and the results showed that the antibody levelsof the pET28a-HBc-EB group and pET22b-EB-BIg group were significantly higher than the inactivatedvaccine group (P<0.01or P<0.05), the antibody levels of pET28a-HBc-EB group were significantlyhigher than the pET22b-EB-BIg group (P<0.05); The liquid phase blocking antibody titer of thepET22b-EB-BIg group was near vaccine group while pET28a-HBc-EB group higher than inactivatedvaccine group, which was from1:256to1:512. T-lymphocyte proliferation assay was performed usingthe Cell Titer96aqueous one solution assay (MTS assay). The results showed that the stimulation indexof the pET28a-HBc-EB group and pET22b-EB-BIg group were significantly higher than the inactivatedvaccine group (P<0.01). The CD4+and CD8+T cell were detected by the Flow cytometry. The resultsshowed that the CD4+/CD8+ratio of the pET28a-HBc-EB group and the pET22b-EB-Big group wassignificantly higher than the inactivated vaccine group (P<0.01and <0.05). The cytokine ELISA assayshowed that the mouse spleen cells stimulated IL-2and IFN-gamma levels of the pET28a-HBc-EBgroup and pET22b-EB-BIg were significantly higher than those in the inactivated vaccine group(P<0.01); and the IL-4and IL-10levels of the pET28a-HBc-EB group were significantly higher than theinactivated vaccine group (P<0.05), while the difference between the pET22b-EB-BIg group and theinactivated vaccine was not significant. In conclusion, the Asia1FMDV type multi-epitopeimmunogenic vaccines had the good immune effect, which stimulate the mice to generate high levels ofhumoral and cellular immunity, especially the pET28a-HBc-EB immunogen.
(3) Production of multi-epitope immunogen in Pichia pastoris and effects of immunity by oral
1) Production of multi-epitope immunogen in Pichia pastoris
In this study, as the Pichia pastoris has the characteristics of the eukaryotic cell expression systemmodification, we select Pichia pastoris to generate multi-epitope immunogen that has the natural structure. The multi-epitopes gene EB was digested from pET28a-HBc-EB and cloned into pPICZA, toconstruct the recombinant expression plasmid pPICZA-HBc-EB. Using the plasmids containing Asia1FMDV gene prepared in our lab as the template, the genes of the three proteins were cloned into Pichiapastoris expression vector-pPICZA. Then the restriction enzyme sites were introduced into theexpression cassette individually by PCR, which was ligated into pPICZA orderly to construct thepPICZA-vp031. Western blot and Dot ELISA showed that the recombinant expression products hadgreat antigenicity.
2) Effects of immunity by oral
The mice were immunized by oral with recombinant Pichia pastoris at the dose of5×108. Theresults showed that the antibody levels of the experimental group pPICZA-HBc-EB and pPICZA-vp031were significantly higher than pPICZA control group at28d post immunization (P<0.01), butsignificantly lower than inactivated vaccine group (P<0.01); the liquid phase blocking antibody titer was1:8to1:16. The mucosal sIgA levels of flushing fluid of the mice immunized with Pichia pastoris weresignificantly higher than inactivated vaccine group (P<0.01); and the mucosa-specific sIgA of theexperimental group pPICZA-HBc-EB and pPICZA-vp031were significantly higher than inactivatedvaccine group, pPICZA group (P<0.01). The stimulation index of pPICZA-HBc-EB group andpPICZA-vp031group were significantly higher than the inactivated vaccine group (P<0.01) and thepPICZA-HBc-EB group was higher than pPICZA-vp031group (P<0.05). The CD4+/CD8+ratio of theinactivated vaccine group were significantly higher than pPICZA-HBc-EB group and pPICZA-vp031group (P <0.01). The cytokine ELISA assay showed that the mouse spleen cells stimulated IL-2andIFN-gamma levels of pPICZA-HBc-EB group were significantly higher than those in the vaccine group(P<0.01), while the differences among the pPICZA-vp031group the inactivated vaccine group were notsignificant (P>0.05); the IL-4and IL-10levels of pPICZA-HBc-EB group and pPICZA-vp031grouphad no significant difference with the inactivated vaccine group (P>0.05). Taken together, the resultsindicated that the oral immunization with the Asia1oral immunogen could not only stimulate theproduction of humoral and cellular immunity, but also significantly induce mucosal immunity.
(4) Effects of immunity by intranasal
The mice were immunized i.n. with the recombinant protein pET28a-HBc-EB andpET22b-EB-BIg.28d past first immunization, results showed that the antibody levels of thepET22b-EB-BIg group were significantly higher than the pET28a-HBc-EB group (P<0.01),significantly lower than the inactivated vaccine group (P<0.01), with lower the antibody grown trend;The liquid phase blocking antibody titer was down to1:8; However, the level of total sIgA and specificsIgA of the pET22b-EB-BIg group were significantly higher than the other groups (P<0.01); Thestimulation index of the pET22b-EB-BIg group was significantly higher than the other groups (P<0.05);While, its CD4+/CD8+ratio was significantly lower than inactivated vaccine group (P<0.01). Theseresults indicate that Bovine IgG2a Fc fragment fusion protein could stimulate both the humoral and cellular immune responses, which can induce mucosal immunity, although the overall strength is nothigh, while pET28a-HBc-EB did not, our work confirmed for the first time the feasibility of targetingthe FcRn stimulate mucosal immune, which may provide a new way for preparing the FMD mucosalvaccine.
机体抵抗口蹄疫病毒（Foot-and-mouth Disease Virus，FMDV）感染过程中，依赖于中和抗体的体液免疫反应是最主要的。虽然灭活疫苗免疫能够产生高水平的中和抗体，但高水平的中和抗体并不是总能为机体提供完全的保护，同时CD4+T细胞在诱导机体体液抗体水平方面发挥着重要的作用，提示同时提高机体的体液免疫和细胞免疫对于FMD的预防更有意义。复合表位疫苗减少了疫苗中与免疫无关的成分，同时可对T、B细胞抗原表位进行不同组合，是实现口蹄疫体液免疫应答和细胞免疫应答的理想选择。FMDV主要是依靠接触和空气进行传播，黏膜免疫应答能够在黏膜部位产生大量的分泌性的sIgA，分泌性的sIgA不仅可以在第一时间中和病毒，而且可通过黏膜的方式进一步激活系统免疫反应，进而阻止病毒的侵入。所以有效的诱导黏膜部位产生sIgA是口蹄疫疫苗发展的新方向。
本研究人工设计了复合表位肽，并分别以乙型肝炎病毒核心抗原（HBcAg）和牛IgG2a Fc片段为载体，利用原核表达系统和毕赤酵母表达系统，制备三种复合表位免疫原pET28a-HBc-EB、pET22b-EB-BIg和rGS115-pPICZA-HBc-EB，通过腹腔注射、口服和滴鼻三种途径分别对小鼠进行免疫，从体液免疫、细胞免疫和黏膜免疫水平对复合表位疫苗的免疫效果进行综合评价。
（1）Asia1型FMDV复合表位免疫原的设计及合成
选取VP1上第140~160位、第200~211位两个B细胞表位和VP1上第20~40位、3A上第21~35两个T细胞表位为候选表位，并在其间引入合适的linker序列，构成了Asia1型FMDV的复合表位。该复合表位肽通过linker序列GSGSG融合于HBcAg的第78~79位氨基酸（刺突位置），软件分析表明，Linker序列在表位之间形成柔性区域和转角，特别是序列（G4S）3的引入保证了两个B细胞表位的独立作用，而复合表位肽所在的HBc刺突位置具有较高的抗原性且大部分存在于表位部分，并有出现在分子表面的可能性，将融合有复合表位的HBcAg序列优化后合成。
（2）复合表位免疫原在原核表达系统中的制备及腹腔免疫效果评价
1)复合表位免疫原在原核表达系统中的制备
以合成的质粒pUC57-simple-HBc-EB为模板，PCR扩增出目的基因，产物酶切后与pET28a连接，得到重组表达质粒pET28a-HBc-EB。以合成的含有牛IgG2a Fc片段基因的pUC57-simple-BIg质粒为模板，扩增出牛IgG2a Fc片段基因，并利用融合PCR方法与复合表位基因连接，酶切后与pET22b连接，得到重组表达质粒pET22b-EB-BIg。复性后的重组pET28a-HBc-EB在电镜下可观察到病毒样粒子的存在，而重组蛋白pET22b-EB-BIg也形成了类似免疫球蛋白单体的形式，能够与兔抗牛抗体直接结合。Western blot鉴定结果表明两免疫原均具备良好的抗原性，更为重要的是Dot ELISA结果显示复性后的复合表位免疫原能够与Asia1型FMDV VP1构象表位抗体很好的结合，间接说明复合后的表位免疫原能够一定程度上折叠为类似于Asia1型VP1蛋白构象型表位的抗原结构。
2)复合表位免疫原腹腔免疫效果评价
免疫后28d的检测结果表明，pET28a-HBc-EB组和pET22b-EB-BIg组小鼠抗体水平显著高于疫苗免疫组（P＜0.01或P＜0.05），pET28a-HBc-EB组的抗体水平显著高于pET22b-EB-BIg组（P＜0.05）；pET28a-HBc-EB组液相阻断抗体效价高于疫苗免疫组，为1:256~1:512；而pET22b-EB-BIg组液相阻断抗体效价为1:128~1:256，与灭活疫苗组相当；pET28a-HBc-EB组和pET22b-EB-BIg组的淋巴细胞刺激指数显著高于灭活疫苗免疫组（P＜0.01）。pET28a-HBc-EB组和pET22b-EB-BIg组的CD4+/CD8+比值显著高于灭活疫苗组（P＜0.01，P＜0.05）。pET28a-HBc-EB组和pET22b-EB-BIg组小鼠脾细胞刺激后产生的IL-2和IFN-γ显著高于灭活疫苗组（P＜0.01），pET28a-HBc-EB组IL-4和IL-10产生水平显著高于灭活疫苗组（P＜0.05），而pET22b-EB-BIg组与灭活疫苗组无显著差异。表明Asia1型FMDV多表位免疫原具有良好的免疫效果，既可刺激机体产生高水平的体液免疫，又可以刺激机体发生细胞免疫，其中以pET28a-HBc-EB组免疫效果最佳。
复合表位免疫原腹腔免疫较为理想的效果证实了复合表位免疫原的有效性，为口服免疫和滴鼻免疫提供了保证。
（3）复合表位免疫原在毕赤酵母表达系统中的制备及口服免疫效果评价
1)复合表位免疫原在毕赤酵母表达系统中的制备
本研究鉴于毕赤酵母具备真核细胞表达系统的修饰功能的特点，以期获得具有天然结构的复合表位免疫原。酶切重组质粒pET28a-HBc-EB，获得复合表位多肽基因，与pPICZA胞内表达载体连接，获得重组质粒pPICZA-HBc-EB；以本实验保存的含有Asia1型FMDV全基因的质粒为模板，分别扩增病毒衣壳蛋白编码基因，并分别克隆于毕赤酵母表达载体pPICZA中，然后通过PCR方法在含有各表达盒两端引入酶切位点，各表达盒经酶切后依次将连入pPICZA载体中，最终构建酵母表达质粒pPICZA-vp031；Western blot和Dot ELISA鉴定结果表明表达产物具有良好的抗原性。
2)重组毕赤酵母以5×108CFU剂量口服免疫小鼠，免疫后28d检测结果表明，pPICZA-HBc-EB组和pPICZA-vp031组抗体水平显著低于灭活疫苗免疫组（P＜0.01）；液相阻断抗体效价为1:8~1:16；毕赤酵母免疫组小鼠黏膜冲洗液sIgA和特异性sIgA总量显著高于疫苗组（P＜0.01）；pPICZA-HBc-EB组和pPICZA-vp031组的黏膜特异性sIgA显著高于灭活疫苗组（P＜0.01）；pPICZA-HBc-EB组和pPICZA-vp031组淋巴细胞增殖指数均显著高于灭活疫苗免疫组（P＜0.01），而pPICZA-HBc-EB组显著高于pPICZA-vp031组（P＜0.05）；灭活疫苗组CD4+/CD8+比值显著高于pPICZA-HBc-EB组和pPICZA-vp031组（P＜0.01）；pPICZA-HBc-EB组小鼠脾细胞刺激后产生的IL-2和IFN-γ显著高于灭活疫苗组（P＜0.01），而pPICZA-vp031组则与灭活疫苗组无显著差异（P＞0.05），pPICZA-HBc-EB组和pPICZA-vp031组IL-4和IL-10水平与灭活疫苗组无显著差异（P＞0.05）。说明口服重组酵母菌不仅可刺激机体产生体液免疫和细胞免疫，而且还能显著地诱导机体黏膜免疫。证实了复合表位免疫原通过口服免疫激发黏膜免疫的可行性。
（4）复合表位免疫原滴鼻免疫效果的评价
利用原核系统制备的复合表位疫苗对小鼠进行滴鼻免疫，免疫后28d pET22b-EB-BIg组抗体水平显著高于pET28a-HBc-EB组（P＜0.01），显著低于灭活疫苗免疫组（P＜0.01），且抗体上升趋势较为缓慢；液相阻断抗体效价低于1:8；pET22b-EB-BIg滴鼻免疫组黏膜冲洗液总sIgA水平、黏膜特异性sIgA抗体水平显著高于灭活疫苗组、pET28a-HBc-EB滴鼻免疫组（P＜0.01）；pET22b-EB-BIg滴鼻免疫组的刺激指数显著高于其他各组（P＜0.05）；pET22b-EB-BIg滴鼻免疫组CD4~+/CD8~+比值显著低于灭活疫苗免疫组（P＜0.01）。上述结果说明pET22b-EB-BIg免疫后小鼠上支气管和肺部冲洗液、肠冲洗液和生殖道冲洗液中均有特异性sIgA的分泌，同时机体产生一定的体液免疫和细胞免疫，而pET28a-HBc-EB则无此效应，首次实践了靶向FcRn激发黏膜免疫的可行性，为FMD黏膜疫苗的制备提供一种新的思路。
Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral diseaseof cloven-hoofed animals worldwide. Thus, controlling the disease especially caused by the O, A andAsia1foot and mouth disease virus (FMDV) in China through regular vaccination and slaughter ofinfected and contact animals are very important. Currently, the conventional FMD inactivated vaccinehas played an important role in controlling the disease.
Humoral immune response of neutralizing antibodies plays an important role in the anti-FMDVprocess. Production of high titres of neutralizing antibodies induced by the inactivated vaccine is notalways protective in livestock. Stimulating CD4+T-cell responses is important for the induction ofoptimum antibody responses to inactivated vaccine. Therefore, it is essential to consider the efficacy andpractical use of vaccines in order to induce an effective humoral immune response and cell-mediatedimmunity. The epitope vaccines combined with the T cell and B cell epitopes, which reduced theingredients independent within the vaccines is the ideal choice to achieve high humoral and cellularimmune response. The spread of FMD is mainly based on the close contact and the air. The mucosalimmunity induces the mucous membrane to produce many sIgA, which not only can neutralize the virusat the first time, but also induce system immune response. Therefore, effective in inducing the mucousmembranes sIgA is the new direction of development of the foot-and-mouth disease vaccine.
In this study, a multi-epitope peptide was designed, fused to HBcAg and Bovine IgG2a Fc,individually. The three multi-epitope immunogens pET28a-HBc-EB, pET22b-EB-BIg andrGS115-pPICZA-HBc-EB were expressed in E.coli and Pichia pastoris. The three multi-epitopeimmunogens were used to immune mice by intraperitoneal injection, oral and intranasal, then theimmune effects were evaluated by humoral immunity, cellular immunity and mucosal immunity.
(1) Design and synthesis of Asia1FMDV multi-epitope immunogen
In this study, two B cell epitopes (141-160aa and200-213aa) in VP1protein and the T cell epitopes(21-40aa in VP1and21-35aa in3A protein) were linked by the suitable linker sequence between themand synthesized as the candidate contents of the vaccine (the multi-epitopes designated as the EB). ThisEB sequences were inserted to the78to79amino acids of the HBcAg sequence with the linker GSGSG(spike position), and the multi-epitopes designated as the HBC-EB. Based on the software analysis, thelinker sequence formed the flexible regions and corners between the epitopes, especially the sequence(G4S)3guaranteed the two B cell epitopes exist as the independent role. The HBc-EB had a high antigenicity and most of the multi-epitope expressing in the HBc spike position, which was possible insurface. Then, the HBC-EB with its optimized gene sequences was optimized and synthesized.
(2) Generation of multi-epitope immunogen and effects of immunity by intraperitoneal injection
1) Generation of multi-epitope immunogen in E.coli
The target gene was amplified by the PCR and the recombinant expression plasmidpET28a-HBc-EB was constructed. Based on the synthesized pUC57-simple-BIg plasmid as the template,the BIg gene was amplified and fused by the EB gene to generate the recombinant expression plasmidpET22b-EB-BIg. The recombinant plasmids were expressed in E.coli. Electron microscopy showed thatthe refolded protein pET28a-HBc-EB complex formed as virus-like particles. Western blot and DotELISA results confirmed that the recombinant proteins had great antigenicity, and the recombinantprotein pET22b-EB-BIg reacted directly with Horseradish peroxidase-conjugated rabbit anti-bovine IgGantibody. The immunization strategy was done using the antigens based on the Asia1FMDVmulti-epitope immunogen.
2) Effects of immunity by intraperitoneal injection
The test was taken at28d after the mice immunized and the results showed that the antibody levelsof the pET28a-HBc-EB group and pET22b-EB-BIg group were significantly higher than the inactivatedvaccine group (P<0.01or P<0.05), the antibody levels of pET28a-HBc-EB group were significantlyhigher than the pET22b-EB-BIg group (P<0.05); The liquid phase blocking antibody titer of thepET22b-EB-BIg group was near vaccine group while pET28a-HBc-EB group higher than inactivatedvaccine group, which was from1:256to1:512. T-lymphocyte proliferation assay was performed usingthe Cell Titer96aqueous one solution assay (MTS assay). The results showed that the stimulation indexof the pET28a-HBc-EB group and pET22b-EB-BIg group were significantly higher than the inactivatedvaccine group (P<0.01). The CD4+and CD8+T cell were detected by the Flow cytometry. The resultsshowed that the CD4+/CD8+ratio of the pET28a-HBc-EB group and the pET22b-EB-Big group wassignificantly higher than the inactivated vaccine group (P<0.01and <0.05). The cytokine ELISA assayshowed that the mouse spleen cells stimulated IL-2and IFN-gamma levels of the pET28a-HBc-EBgroup and pET22b-EB-BIg were significantly higher than those in the inactivated vaccine group(P<0.01); and the IL-4and IL-10levels of the pET28a-HBc-EB group were significantly higher than theinactivated vaccine group (P<0.05), while the difference between the pET22b-EB-BIg group and theinactivated vaccine was not significant. In conclusion, the Asia1FMDV type multi-epitopeimmunogenic vaccines had the good immune effect, which stimulate the mice to generate high levels ofhumoral and cellular immunity, especially the pET28a-HBc-EB immunogen.
(3) Production of multi-epitope immunogen in Pichia pastoris and effects of immunity by oral
1) Production of multi-epitope immunogen in Pichia pastoris
In this study, as the Pichia pastoris has the characteristics of the eukaryotic cell expression systemmodification, we select Pichia pastoris to generate multi-epitope immunogen that has the natural structure. The multi-epitopes gene EB was digested from pET28a-HBc-EB and cloned into pPICZA, toconstruct the recombinant expression plasmid pPICZA-HBc-EB. Using the plasmids containing Asia1FMDV gene prepared in our lab as the template, the genes of the three proteins were cloned into Pichiapastoris expression vector-pPICZA. Then the restriction enzyme sites were introduced into theexpression cassette individually by PCR, which was ligated into pPICZA orderly to construct thepPICZA-vp031. Western blot and Dot ELISA showed that the recombinant expression products hadgreat antigenicity.
2) Effects of immunity by oral
The mice were immunized by oral with recombinant Pichia pastoris at the dose of5×108. Theresults showed that the antibody levels of the experimental group pPICZA-HBc-EB and pPICZA-vp031were significantly higher than pPICZA control group at28d post immunization (P<0.01), butsignificantly lower than inactivated vaccine group (P<0.01); the liquid phase blocking antibody titer was1:8to1:16. The mucosal sIgA levels of flushing fluid of the mice immunized with Pichia pastoris weresignificantly higher than inactivated vaccine group (P<0.01); and the mucosa-specific sIgA of theexperimental group pPICZA-HBc-EB and pPICZA-vp031were significantly higher than inactivatedvaccine group, pPICZA group (P<0.01). The stimulation index of pPICZA-HBc-EB group andpPICZA-vp031group were significantly higher than the inactivated vaccine group (P<0.01) and thepPICZA-HBc-EB group was higher than pPICZA-vp031group (P<0.05). The CD4+/CD8+ratio of theinactivated vaccine group were significantly higher than pPICZA-HBc-EB group and pPICZA-vp031group (P <0.01). The cytokine ELISA assay showed that the mouse spleen cells stimulated IL-2andIFN-gamma levels of pPICZA-HBc-EB group were significantly higher than those in the vaccine group(P<0.01), while the differences among the pPICZA-vp031group the inactivated vaccine group were notsignificant (P>0.05); the IL-4and IL-10levels of pPICZA-HBc-EB group and pPICZA-vp031grouphad no significant difference with the inactivated vaccine group (P>0.05). Taken together, the resultsindicated that the oral immunization with the Asia1oral immunogen could not only stimulate theproduction of humoral and cellular immunity, but also significantly induce mucosal immunity.
(4) Effects of immunity by intranasal
The mice were immunized i.n. with the recombinant protein pET28a-HBc-EB andpET22b-EB-BIg.28d past first immunization, results showed that the antibody levels of thepET22b-EB-BIg group were significantly higher than the pET28a-HBc-EB group (P<0.01),significantly lower than the inactivated vaccine group (P<0.01), with lower the antibody grown trend;The liquid phase blocking antibody titer was down to1:8; However, the level of total sIgA and specificsIgA of the pET22b-EB-BIg group were significantly higher than the other groups (P<0.01); Thestimulation index of the pET22b-EB-BIg group was significantly higher than the other groups (P<0.05);While, its CD4+/CD8+ratio was significantly lower than inactivated vaccine group (P<0.01). Theseresults indicate that Bovine IgG2a Fc fragment fusion protein could stimulate both the humoral and cellular immune responses, which can induce mucosal immunity, although the overall strength is nothigh, while pET28a-HBc-EB did not, our work confirmed for the first time the feasibility of targetingthe FcRn stimulate mucosal immune, which may provide a new way for preparing the FMD mucosalvaccine.
引文
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