College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, Heilongjiang, PR China; Heilongjiang Key Laboratory for Experimental Animals and Comparative Medicine, Harbin 150030, Heilongjiang, PR China.
College of Food Science, Northeast Agricultural University, Harbin 150030, Heilongjiang, PR China.
Poult Sci. 2020 Jun;99(6):2967-2975. doi: 10.1016/j.psj.2020.03.014. Epub 2020 Apr 9.
Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection.
禽球虫病给全球家禽养殖业造成了巨大的经济损失。探索针对球虫病的新概念疫苗已逐渐成为研究热点。本研究以从鸡肠道中分离到的一株性能优异的屎肠球菌（MDXEF-1）为载体，递呈柔嫩艾美耳球虫靶蛋白。将携带柔嫩艾美耳球虫 NAΔ3-1E 基因（不含起始密码子 ATG 和终止密码子 TAA 的 3-1E 蛋白编码基因）的树突状细胞靶向肽（DCpep）融合蛋白的质粒 pTX8048-SP-DCpep-NAΔ3-1E-CWA 电转化至 MDXEF-1 中，构建重组菌 MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA，使 3-1E 蛋白通过细胞壁锚定（CWA）序列共价锚定在细菌细胞表面。通过 Western blot 检测目的融合蛋白 DCpep-NAΔ3-1E-CWA 的表达。每只鸡间隔 2 周用表达 DCpep-NAΔ3-1E 融合蛋白的活屎肠球菌（DCpep-NAΔ3-1E 组）、表达 NAΔ3-1E 蛋白的活屎肠球菌（NAΔ3-1E 组）和仅含空载体的活屎肠球菌（对照组）进行 3 次免疫。免疫后，用同源的柔嫩艾美耳球虫孢子化卵囊攻毒，评估各组的免疫反应和保护效果。结果显示，与 NAΔ3-1E 组相比，DCpep-NAΔ3-1E 组鸡血清 IgG 水平、盲肠冲洗液分泌型 IgA 水平、外周血 CD4+和 CD8α+细胞比例以及脾脏 IL-2 和 IFN-γ mRNA 表达水平均显著升高（P<0.05）。与表达 NAΔ3-1E 蛋白的活屎肠球菌相比，口服免疫表达 DCpep-NAΔ3-1E 的活屎肠球菌对同源攻毒具有更高的保护效力，包括体重显著增加、卵囊减少率增加和盲肠平均病变评分降低。这些结果表明，表达柔嫩艾美耳球虫 3-1E 蛋白的树突状细胞靶向肽融合蛋白的重组屎肠球菌可能是预防艾美耳球虫感染的一种有潜力的方法。
