壳寡糖对大熊猫轮状病毒VP6-VP7亚单位疫苗免疫作用的研究

doi:10.3969/j.issn.1004-1524.2019.12.08

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (12): 2005-2010.DOI: 10.3969/j.issn.1004-1524.2019.12.08

壳寡糖对大熊猫轮状病毒VP6-VP7亚单位疫苗免疫作用的研究

文继锋, 申欢欢, 阴文奇, 龚永平, 易可可, 邓英, 颜其贵^*

四川农业大学动物医学院,四川成都 611130

收稿日期:2019-03-01 出版日期:2019-12-25 发布日期:2019-12-25
通讯作者: 颜其贵,E-mail:yanqigui@126.com,13981608208
作者简介:文继锋(1991-),男,湖北恩施人,苗族,硕士研究生,主要从事兽医微生物与免疫学研究。E-mail:314690952@qq.com
基金资助:
成都大熊猫繁育研究基金会项目(CPF研2012-12,CPF研2017-35)

Research on immunological effect of chitosan oligosaccharide on subunit vaccine of giant panda rotavirus VP6-VP7 protein

WEN Jifeng, SHEN Huanhuan, YIN Wenqi, GONG Yongping, YI Keke, DENG Ying, YAN Qigui^*

College of Veterinary Medicine, Sichuan Agricultural University, Chendu 611130, China

Received:2019-03-01 Online:2019-12-25 Published:2019-12-25

摘要/Abstract

摘要： 为研究壳寡糖(COS)对大熊猫轮状病毒(GPRV)重组蛋白VP6-VP7的免疫增强作用,以原核表达的重组VP6-VP7蛋白为抗原,添加浓度为2.0 mg·mL^-1 COS作为免疫佐剂制成GPRV VP6-VP7-COS亚单位疫苗。选取SPF级昆明小鼠78只随机分为4组。PC组免疫纯化的重组VP6-VP7蛋白,A组免疫VP6-VP7-COS疫苗,B组免疫VP6-VP7氢氧化铝胶佐剂疫苗,NC组注射PBS(对照组)。在0、15、30 d进行免疫接种,每次接种完成后于每周每组随机挑选6只小鼠采血分离血清,分别检测小鼠血清IgG、IgA抗体效价、T淋巴细胞转化率、细胞因子的含量,评估COS对GPRV重组VP6-VP7蛋白诱导的免疫应答反应的调节作用。试验结束后,取各组小鼠注射部位的肌肉组织进行病理组织学分析,以评价COS作为免疫佐剂的安全性。结果表明,VP6-VP7-氢氧化铝胶佐剂组、VP6-VP7-COS组免疫小鼠血清中所产生的IgG均显著(P<0.05)高于其他各组。VP6-VP7-COS组小鼠产生的IgA抗体含量显著(P<0.05)高于其他各组,VP6-VP7-氢氧化铝胶佐剂组和VP6-VP7-COS物理混合组诱导T淋巴细胞增殖的能力显著(P<0.05)高于VP6-VP7蛋白组。VP6-VP7-COS组、VP6-VP7-氢氧化铝胶佐剂组中IL-2、IL-4、IL-5及IFN-γ的含量均显著(P<0.05)高于其他组,组织病理学分析结果表明,以COS作为佐剂的VP6-VP7亚单位疫苗免疫小鼠后注射部位的肌肉组织未出现病理变化。本研究表明,GPRV重组蛋白VP6-VP7能够显著诱导动物机体的免疫应答,壳寡糖对GPRV VP6-VP7蛋白呈现较好的免疫增强效果。本研究为研制安全、无副作用和高免疫保护力的GPRV 亚单位疫苗提供了参考。

关键词: 大熊猫轮状病毒, VP6-VP7蛋白, 壳寡糖, 免疫增强作用, 亚单位疫苗

Abstract: To research the immunopotentiation effect of chitosan oligosaccharide on the giant panda rotavirus (GPRV) recombinant protein VP6-VP7, prokaryotic expressed recombinant VP6-VP7 protein were used as antigen and added with 2.0 mg·mL^-1 chitosan oligosaccharide as an immunoadjuvant to make a GPRV VP6-VP7-COS subunit vaccine.78 SPF Kunming mice were randomly divided into 4 groups. Group PC immunopurified recombinant VP6-VP7 protein, group A immunized VP6-VP7-COS subunit vaccine, group B immunized VP6-VP7 aluminum hydroxide gel adjuvant subunit vaccine, group NC injected with PBS. At 0, 15 and 30 days, immunization was carried out. After each vaccination, 6 mice were randomly selected from each group to collect serum, and serum IgG, IgA antibody titer, T lymphocyte transformation rate and cytokine content were measured. Muscle tissues of the injection site of each group were subjected to histopathological analysis to evaluate the toxic side effects of COS as an immunoadjuvant. The results showed that the IgG level of VP6-VP7-aluminum hydroxide gel adjuvant group and VP6-VP7-COS group were significantly (P<0.05) higher than the other groups. The IgA level in the VP6-VP7-COS group was significantly (P<0.05) higher than that in the other groups. The ability of VP6-VP7-aluminum hydroxide gel adjuvant group and VP6-VP7-COS physical mixing group to induce T lymphocyte proliferation were significantly (P<0.05)higher than that of VP6-VP7 protein group. IL-2, IL-4, IL-5 and IL-γ in VP6-VP7-COS group and VP6-VP7-aluminum hydroxide gel adjuvant group were significantly (P<0.05) higher than the other groups. Histopathological analysis showed that there were no pathological changes in the muscle tissue of the injected site of VP6-VP7 subunit vaccine immunized with COS as adjuvant. This study showed that GPRV recombinant protein VP6-VP7 can significantly induce the immune response in animals, and chitosan oligosaccharide had a better immune enhancement effect on GPRV VP6-VP7 protein. This study provided a reference for the development of safe, non-side-effect and high immunoprotective GPRV subunit vaccines.

Key words: giant panda rotavirusv, VP6-VP7 protein, chitosan oligosaccharide, immune enhancement, subunit vaccine

中图分类号:

S858.28

文继锋, 申欢欢, 阴文奇, 龚永平, 易可可, 邓英, 颜其贵. 壳寡糖对大熊猫轮状病毒VP6-VP7亚单位疫苗免疫作用的研究[J]. 浙江农业学报, 2019, 31(12): 2005-2010.

WEN Jifeng, SHEN Huanhuan, YIN Wenqi, GONG Yongping, YI Keke, DENG Ying, YAN Qigui. Research on immunological effect of chitosan oligosaccharide on subunit vaccine of giant panda rotavirus VP6-VP7 protein[J]. , 2019, 31(12): 2005-2010.

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壳寡糖对大熊猫轮状病毒VP6-VP7亚单位疫苗免疫作用的研究

Research on immunological effect of chitosan oligosaccharide on subunit vaccine of giant panda rotavirus VP6-VP7 protein

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