丝状支原体山羊亚种LppA蛋白N末端基因真核表达载体构建及小鼠免疫效果分析

doi:10.11843/j.issn.0366-6964.2022.03.017

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (3): 847-856.doi: 10.11843/j.issn.0366-6964.2022.03.017

丝状支原体山羊亚种LppA蛋白N末端基因真核表达载体构建及小鼠免疫效果分析

尹德晶¹, 吴燕¹, 岳筠³, 杨鹏¹, 陈静¹, 王慧³, 张双翔³, 王开功^1,2*, 程振涛^1,2*

1. 贵州大学动物科学学院, 贵阳 550025;
2. 贵州省动物疫病与兽医公共卫生重点实验室, 贵阳 550025;
3. 贵州省动物疫病预防控制中心, 贵阳 550008

收稿日期:2021-07-30 出版日期:2022-03-23 发布日期:2022-03-31
通讯作者: 王开功,主要从事动物疫病预防研究,E-mail:kgwang@gzu.edu.cn;程振涛,主要从事动物病原病理学研究,E-mail:chengzhentao@sohu.com
作者简介:尹德晶(1997-),女,贵州六盘水人,硕士生,主要从事动物疫病研究,E-mail:654625128@qq.com
基金资助:
国家自然科学基金项目(31660723);国家自然科学基金项目(32060786);贵州省科技计划项目(黔科合基础[2019]1181号);贵州省科技计划项目(黔科合支撑[2021]一般161号);贵州省动物疫病预防控制中心人才基地建设项目(黔疫控[2019]06号)

Construction of Eukaryotic Expression Vector of N-terminal Gene of LppA Protein of Mycoplasma Mycoides subsp.Capri and Analysis of Its Immune Efficacy in Mice

YIN Dejing¹, WU Yan¹, YUE Jun³, YANG Peng¹, CHEN Jing¹, WANG Hui³, ZHANG Shuangxiang³, WANG Kaigong^1,2*, CHENG Zhentao^1,2*

1. College of Animal Science, Guizhou University, Guiyang 550025, China;
2. Guizhou Key Laboratory of Animal Disease and Veterinary Public Health, Guiyang 550025, China;
3. Guizhou Center for Animal Disease Prevention and Control, Guiyang 550008, China

Received:2021-07-30 Online:2022-03-23 Published:2022-03-31

摘要/Abstract

摘要： 基于丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri, Mmc)贵州株的LppA蛋白N末端基因,构建真核重组表达质粒pVAX1-LppA,并对免疫效果进行分析,为防控羊支原体肺炎提供新思路。以构建的真核重组表达质粒pVAX1-LppA(50、100、150 μg)、pVAX1空载体、无菌PBS分别免疫小鼠。通过ELISA方法检测小鼠血清中抗体和细胞因子(IL-2、IFN-γ、IL-4)水平,MTT法检测脾淋巴细胞增殖情况,流式细胞术检测CD4⁺、CD8⁺ T淋巴细胞占总细胞数比例的变化,攻毒试验评估对小鼠的保护效率,并采集小鼠肺制备切片以观察病理变化。结果显示:与空载体pVAX1组及PBS对照组相比,重组质粒pVAX1-LppA组小鼠体内的抗体和细胞因子(IL-2、IFN-γ、IL-4)水平显著升高,脾细胞增殖能力更强,CD4⁺、CD8⁺ T淋巴细胞占总细胞数的比例显著增多,对小鼠具有一定的攻毒保护能力,肺部病理损伤明显减轻,发病动物数量减少,其中100 μg的重组质粒pVAX1-LppA保护率最高,为80%。综上表明,LppA蛋白具有良好的免疫原性,重组质粒pVAX1-LppA能激活强烈的体液免疫和细胞免疫应答,可作为羊支原体肺炎的候选疫苗。

关键词: 丝状支原体山羊亚种, LppA蛋白, DNA疫苗, 免疫效果

Abstract: Based on the N-terminal gene of LppA protein of Mycoplasma mycoides subsp. capri (Mmc) Guizhou strain, the eukaryotic recombinant expression plasmid pVAX1-LppA was constructed, and the immune effect was analyzed to provide new ideas for the prevention and control of mycoplasmal pneumonia of sheep and goats (MPSG). Mice were immunized with the constructed eukaryotic recombinant expression plasmid pVAX1-LppA (50, 100, 150 μg), pVAX1 empty vector, and PBS, respectively. The levels of antibodies and cytokines (IL-2, IFN-γ and IL-4) in sera of mice were detected by ELISA. The proliferation of splenic lymphocytes was detected by MTT assay and the ratio of CD4⁺, CD8⁺ T lymphocytes to total cells was detected by flow cytometry. The protective efficiency of mice was evaluated by the challenge test. The lungs of mice were collected for preparation of sections to observe the pathological changes. The results showed that compared with the empty vector pVAX1 and PBS control groups, the levels of antibodies and cytokines (IL-2, IFN-γ, IL-4) in mice in the recombinant plasmid pVAX1-LppA group were significantly increased, the proliferation ability of spleen cells was stronger, and the proportion of CD4⁺ and CD8⁺ T lymphocytes in total cells increased significantly. The recombinant plasmid pVAX1-LppA had a certain ability to protect mice from Mmc challenge. The lung pathological damage was significantly reduced, and the number of cases was decreased post immunization. The maximum protection rate of 100 μg recombinant plasmid pVAX1-LppA was 80%. The results showed that LppA protein had good immunogenicity, and the recombinant plasmid pVAX1-LppA could activate strong humoral and cellular immune responses, which could be used as a candidate vaccine for MPSG.

Key words: Mycoplasma mycoides subsp.capri, LppA protein, DNA vaccine, immune effect

中图分类号:

S852.62

尹德晶, 吴燕, 岳筠, 杨鹏, 陈静, 王慧, 张双翔, 王开功, 程振涛. 丝状支原体山羊亚种LppA蛋白N末端基因真核表达载体构建及小鼠免疫效果分析[J]. 畜牧兽医学报, 2022, 53(3): 847-856.

YIN Dejing, WU Yan, YUE Jun, YANG Peng, CHEN Jing, WANG Hui, ZHANG Shuangxiang, WANG Kaigong, CHENG Zhentao. Construction of Eukaryotic Expression Vector of N-terminal Gene of LppA Protein of Mycoplasma Mycoides subsp.Capri and Analysis of Its Immune Efficacy in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 847-856.

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丝状支原体山羊亚种LppA蛋白N末端基因真核表达载体构建及小鼠免疫效果分析

Construction of Eukaryotic Expression Vector of N-terminal Gene of LppA Protein of Mycoplasma Mycoides subsp.Capri and Analysis of Its Immune Efficacy in Mice

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