杀香鱼假单胞菌外膜蛋白Tol C的原核表达及其对大黄鱼的免疫保护效果

Prokaryotic expression of outer membrane protein Tol C of Pseudomonas plecoglossicida and its immunoprotective effect analysis on Larimichthys crocea

摘要:

目的 评价大黄鱼杀香鱼假单胞菌外膜蛋白Tol C (GenBank：EPB97112.1)的免疫保护效果。方法 通过原核表达获得重组的Tol C功能域蛋白，命名为B6。用兔抗杀香鱼假单胞菌多抗血清对B6进行免疫原性的初步检测。将B6与明矾佐剂混合制成亚单位疫苗，腹腔注射免疫大黄鱼，免疫4周后攻毒，检测相对保护率 (relative percent survival，RPS)。同时，采集免疫后第2、4、6、8周的大黄鱼血清、头肾和脾脏，测定血清抗体效价和免疫相关基因的表达变化。采集健康和攻毒后大黄鱼的头肾和脾脏组织，制成石蜡切片，观察组织病理变化。结果 B6分子质量为22 ku。Western blot结果显示，杀香鱼假单胞菌兔抗血清与B6具有强结合条带。接种B6亚单位疫苗4周后，RPS达72.22%。ELISA结果显示，免疫后第2周起，血清抗体效价显著升高，第4周时达到峰值 (log₂ 8.65)，随后略有下降，第8周时降至log₂ 6.98。荧光定量PCR (RT-PCR) 结果显示，免疫后第2、4、6、8周，头肾和脾脏中免疫相关基因均有不同程度的表达上调，其中IL-1β和MHC Ⅰ α的上调幅度最大，头肾中分别达到93.33倍和77.02倍，脾脏中分别达到241.13倍和131.95倍。除CD8外，IgM、IL-1β、CD4、MHC Ⅰ α和MHC Ⅱ β在脾脏中的表达上调幅度均高于头肾。石蜡切片显示，对照组在攻毒后头肾和脾脏组织中出现严重的出血点和结节，并伴随不同程度的铁黄素沉积，B6免疫组在头肾和脾脏组织中仅有少量出血点，无结节和铁黄素沉积。结论 通过对大黄鱼杀香鱼假单胞菌外膜蛋白Tol C进行原核表达和免疫保护效果评价，发现B6具有良好的免疫原性，免疫后4周RPS达72.22 %，具有作为亚单位候选疫苗的潜力。

Abstract:

In recent years, due to the high density of mariculture, the water circulation is not smooth, the sea area's pollution is becoming more and more serious and the disease occurs frequently. Visceral white spot disease caused by Pseudomonas plecoglossicida is a common bacterial disease of Larimichthys crocea in cage culture. A recombinant Tol C domain protein named B6 was obtained by prokaryotic expression to evaluate the immunoprotective effect of Tol C (GenBank: EPB97112.1). The immunogenicity of B6 was preliminarily detected by rabbit anti-P. plecoglossicida polyclonal antiserum. Following this, a subunit vaccine was prepared by mixing B6 with alum adjuvant, and L. crocea was immunized by intraperitoneal injection. The relative protection rate (RPS) was detected 4 weeks after immunization. Meanwhile, the serum, head kidney and spleen of fish were collected at 2, 4, 6 and 8 weeks after immunization, and the serum antibody titer and the expression level of immune-related genes were determined. Finally, the head kidney and spleen of healthy and challenged individuals were both collected and made into paraffin sections to compare histopathological changes. The results showed that the molecular weight of B6 was 22 ku. The Western blot results showed that the rabbit antiserum against P. plecoglossicida could strongly bind to B6 with a single unique band. A total of four weeks after inoculation with B6 subunit vaccine, the RPS reached 72.22%. ELISA showed that the serum antibody titer increased significantly since the second week after immunization, peaked at the fourth week (log₂ value 8.65), and decreased slightly to log₂ value 6.98 in the eighth week. RT-PCR showed that the expression level of immune-related genes in the head kidney and spleen were both up-regulated with varying degrees at 2, 4, 6 and 8 weeks after immunization. Among them, the up-regulation rates of IL-1β and MHCⅠα was the largest, with 93.33 and 77.02 times each in the head kidney, and 241.13 and 131.95 times each in the spleen, respectively. Except for CD8, the expression up-regulation trend of IgM, IL-1β, CD4, MHC Ⅰ α and MHC Ⅱ β in the spleen was all higher than that in the head kidney. Paraffin sections showed that severe bleeding spots and nodules appeared in the head kidney and spleen of the control group after the challenge, accompanied by different degrees of siderophore deposition. Comparatively, there were only a few bleeding spots in the head kidney and spleen of the B6 immunized group, and no nodules and siderophore deposition. In conclusion, our study first reported the prokaryotic expression and immune protection evaluation of the outer membrane protein Tol C from P. plecoglossicida. The results showed the recombinant Tol C protein had strong immunogenicity and good immune protection in L. crocea, with a RPS of 72.22 % 4 weeks after immunization, thus was a potential candidate of subunit vaccine.