建構Lactobacillus plantarum表現豬第二型環狀病毒2d之外殼蛋白用於口服疫苗之開發

Abstract

豬第二型環狀病毒 (porcine circovirus type 2) 感染豬隻所引起各種臨床症狀是目前養豬業面臨到的重大問題。近年來，PCV2d感染豬隻的盛行率有增加的現象。PCV2d中的外殼蛋白 (capsid protein) 可以誘發宿主的免疫反應。Collagen binding gene (Cnb)在Lactobacillus reuteri中可發現，Cnb 的功能可幫助抗原黏附在乳酸菌表面上。本實驗利用益生菌Lactobacillus plantarum 作為載體，成功建構了L. plantarum pLp3050-His6-tag-capsid和L. plantarum pLp3050-cnb-His6-tag-capsid。西方墨點法和流式細胞儀證實兩個不同的重組菌株皆能偵測到capsid蛋白質。相較於未誘導的重組菌株，重組乳酸菌在50 ng/mL SppIP誘導的狀況下，有較慢的生長速率。在質體穩定性試驗中，將L. plantarum pLp3050-His6-tag-capsid接種在無抗生素篩選的培養基下，質體存在率在第72 h仍有82%。將1010 c.f.u的L. plantarum pLp3050-His6-tag-capsid以口服的方式免疫小鼠，在小鼠糞便中可偵測到capsid-specific sIgA抗體產生和中和抗體的產生，且具有中和PCV2的能力。但以1010 c.f.u的 L. plantarum pLp3050-cnb-His6-tag-capsid免疫小鼠，小鼠糞便並不會產生capsid-specific sIgA抗體。不論以L. plantarum pLp3050-His6-tag-capsid和L. plantarum pLp3050-cnb-His6-tag-capsid免疫小鼠，小鼠血液中capsid-specific IgG的含量與控制組相比皆無差異。然而，在免疫L. plantarum pLp3050-His6-tag-capsid和L. plantarum pLp3050-cnb-His6-tag-capsid皆不會促使脾臟細胞分泌IFN-γ，推測以L. plantarum所構築的疫苗，其免疫效應以誘發腸道免疫為主。綜合上述結果，L. plantarum pLp3050-His6-tag-capsid能藉由誘導小鼠黏膜免疫反應產生中和抗體，以降低PCV2感染細胞。

Porcine circovirus type 2 (PCV2) causes severe economic loss in swine industry owing to its pathogenicity to swine. In recent years, the prevalence of PCV2d genotype infections in swine has been increasing. The capsid protein of PCV2d serves as an epitope that can induce host immune response. Collagen binding protein (Cnb), found in Lactobacillus reuteri, can help antigens to anchor on the surface layer of lactic acid bacteria. In this study, we successfully constructed L. plantarum pLp3050-His6-tag-capsid and L. plantarum pLp3050-cnb-His6-tag-capsid. Resutls of Western blot and flow cytometry showed that both recombinant L. plantarum strains can express capsid on the cell wall. The growth curve of 50 ng/mL SppIP-induced L. plantarum pLp3050-His6-tag-capsid is slower than non-induced L. plantarum pLp3050-His6-tag-capsid. The stability experiment shows that pLp3050-His6-tag-capsid existed in 82% of L. plantarum pLp3050-His6-tag-capsid after transformation for 72 h without antibiotics selection. Mice orally immunized with 1010 c.f.u of L. plantarum pLp3050-His6-tag-capsid showed an increased level of capsid-specific sIgA and neutralizing titer in fecal of mice. However, oral immunization 1010 c.f.u of L. plantarum pLp3050-His6-tag-cnb-capsid did not elicit capsid-specific sIgA in mice. In either L. plantarum pLp3050-His6-tag-cnb-capsid or L. plantarum pLp3050-His6-tag-capsid vacinncated mice, no significant differences of specific IgG in circulatioin was displayed as compared with PBS group. The secretion of IFN-γfrom spleenocyte in mice did not increase after boosting both recombinant strains, suggesting the capacity of these probiotic derived engeering vaccine might be restricted in mucosal immune. In conclusion, L. plantarum pLp3050-His6-tag-capsid can induce neutralizing antibody in mice via mucosal immune response to against PCV2 infection.