Division of Health and Applied Sciences, Faculty of Science, Prince of Songkla University, Hatyai, Songkhla, 90110, Thailand.
Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, 80161, Thailand; Research Excellence Center for Innovation and Health Product, Walailak University, Nakhon Si Thammarat, 80161, Thailand.
Dev Comp Immunol. 2022 Oct;135:104459. doi: 10.1016/j.dci.2022.104459. Epub 2022 Jun 1.
Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.
丝氨酸蛋白酶是一类在许多生物系统中具有生物学功能的蛋白水解酶。先前有报道称，副溶血性弧菌的丝氨酸蛋白酶是一种毒力因子。本研究以副溶血性弧菌丝氨酸蛋白酶基因为研究对象，构建了该基因的点突变真核表达质粒 pcDNA3.1-mutVpSP，并将其作为 DNA 疫苗免疫凡纳滨对虾，探讨其作为防治凡纳滨对虾急性肝胰腺坏死病（AHPND）候选疫苗的潜力。结果显示，突变后的丝氨酸蛋白酶基因（mutVpSP）成功构建到 pcDNA3.1 载体上。免疫荧光定位检测发现，该基因在凡纳滨对虾的多种组织中均有转录表达，包括血细胞、肝胰腺、胃、肠、鳃和肌肉等组织。攻毒实验结果表明，mutVpSP 免疫组凡纳滨对虾的累积死亡率为 30%，显著低于对照组的 60%。同时，免疫组对虾的酚氧化酶、血淋巴蛋白、几丁质酶等免疫相关基因的转录水平显著上调。此外，免疫组对虾的体液免疫和细胞免疫水平也得到了显著增强，表现为血清中碱性磷酸酶（AKP）、溶菌酶（LSZ）和抗菌肽（Cecropin）活性升高，吞噬、包埋活性增强，血淋巴中酚氧化酶原（proPO）激活水平升高，肝胰腺中抗菌肽基因转录水平上调，免疫相关组织中免疫反应结节增多。综上所述，该研究结果表明副溶血性弧菌丝氨酸蛋白酶是其重要的毒力因子之一，该基因的点突变体可作为防治 AHPND 的候选 DNA 疫苗。
