猪附红细胞体eno基因重组蛋白的制备及免疫效果研究

doi:10.16431/j.cnki.1671-7236.2020.07.029

摘要/Abstract

摘要： 为了研究猪附红细胞体eno基因重组蛋白的免疫效果，本试验将猪附红细胞体eno基因与pET-15b载体进行连接，转化大肠杆菌BL21感受态细胞。对表达菌株进行诱导表达、纯化，利用纯化后的猪附红细胞体eno重组蛋白（命名为rMseno）对小鼠进行免疫，从而评价该重组蛋白在小鼠体内产生的免疫应答水平。试验将20只4周龄的雄性昆明小鼠随机分为A、B、C、D 4个组。免疫A组接种纯化后的重组蛋白rMseno；免疫B组接种经IPTG诱导表达后的重组菌E.coli-Mseno；对照C组接种等量PBS；对照D组接种未经过诱导表达的重组菌E.coli-Mseno。通过ELISA检测方法分别测定血清中抗猪附红细胞体特异抗体水平及γ干扰素（IFN-γ）和白细胞介素4（IL-4）细胞因子水平，最后通过脾脏淋巴细胞增殖试验来反映小鼠体内T淋巴细胞的增殖情况。结果显示，构建的重组pET-15b-eno质粒目的基因片段大小为1 632 bp，与预期大小相同；纯化的重组蛋白rMseno大小为61 ku，并能够被鼠抗猪附红细胞体血清所识别；免疫A组和免疫B组小鼠血清中抗猪附红细胞体特异抗体水平，IFN-γ、IL-4细胞因子水平以及T淋巴细胞增殖指数均显著或极显著高于对照组（P<0.05；P<0.01）。结果表明，猪附红细胞体eno基因重组蛋白能够诱导小鼠产生较高的体液免疫水平以及细胞免疫应答水平。

关键词: 猪附红细胞体; 重组蛋白纯化; 昆明小鼠; 免疫效果

Abstract: In order to study the immune effect of the recombinant protein of Mycoplasma suis eno gene,the Mycoplasma suis eno gene was linked with pET-15b vector and transformed into E.coli BL21 competent cells.The recombinant Mycoplasma suis eno protein (named rMseno) was used to immunize mice,and the immune response of the recombinant protein in mice was evaluated.Twenty four-week-old male Kunming mice were randomly divided into four groups:Group A,group B,group C and group D.Immune group A was inoculated with recombinant protein rMseno.Immune group B was inoculated with E.coli-Mseno induced by IPTG.Control group C was inoculated with the same amount of PBS.Control group D was inoculated with E.coli-Mseno which was not induced expression.The levels of specific antibody against Mycoplasma suis,IFN-γ and IL-4 cytokines in serum were determined by ELISA,and the proliferation of T lymphocyte in mice was detected by spleen lymphocyte test,the experimental data were analyzed statistically.The results showed that the target gene fragment of the recombinant pET-15b-eno plasmid was 1 632 bp,and the purified recombinant protein rMseno was 61 ku,which could be recognized by the mouse anti-Mycoplasma suis serum.The levels of anti-Mycoplasma suis specific antibody,IFN-γ,IL-4 cytokines and T lymphocyte proliferation index in the sera of mice in immune group A and immune group B were significantly or extremely significantly higher than those in control groups (P<0.05;P<0.01).The results showed that eno gene recombinant protein of Mycoplasma suis could induce high humoral immunity and cellular immune response in mice.

Key words: Mycoplasma suis; purification of recombinant protein; Kunming mice; immune effect

中图分类号:

S858.28

闫宗斌, 伍生军, 杨静, 闫可心, 相思宇, 许应天, 薛书江, 柴方红. 猪附红细胞体eno基因重组蛋白的制备及免疫效果研究[J]. 中国畜牧兽医, 2020, 47(7): 2231-2238.

YAN Zongbin, WU Shengjun, YANG Jing, YAN Kexin, XIANG Siyu, XU Yingtian, XUE Shujiang, CHAI Fanghong. Preparation of Recombinant Protein of Mycoplasma suis eno Gene and Its Immunity Effect[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2231-2238.

参考文献

[1] HOELZLE L E,ZEDER M,FELDER K M,et al.Pathobiology of Mycoplasma suis[J].Veterinary Journal,2014,202(1):20-25.
[2] 邹亚文,贾杏林,黄琳.猪附红细胞体分子检测技术的研究进展[J].黑龙江畜牧兽医,2016,5:84-86. ZOU Y W,JIA X L,HUANG L.Advances in molecular detection of Mycoplasma suis[J].Heilongjiang Animal Science and Veterinary Medicine,2016,5:84-86.(in Chinese)
[3] DIETZ S,LSAAEK C,MACK S L,et al.Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs[J].Proteomics,2016,16(4):609-613.
[4] NEIMARK H,JOHANSSON K E,RIKIHISA Y,et al.Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candidatus Mycoplasma haemofelis’,‘Candidatus Mycoplasma haemomuris’,‘Candidatus Mycoplasma haemosuis’ and ‘Candidatus Mycoplasma wenyonii’[J].International Journal of Systematic and Evolutionary Microbiology,2001,51(Pt3):891-899.
[5] HOELZLE L E.Significance of haemotrophic mycoplasmas in veterinary medicine with particular regard to the Mycoplasma suis infection in swine[J].Berliner and Munchener Tierarztliche Wochenschrift,2007,120(1-2):34-41.
[6] HOELZLE L E.Haemotrophic mycoplasmas:Recent advances in Mycoplasma suis[J].Veterinary Microbiology,2008,130(3-4):215-226.
[7] 唐丽,李晓云,贾杏林.猪附红细胞体分子生物学研究进展[J].中国畜牧兽医,2013,40(7):81-83. TANG L,LI X Y,JIA X L.Research progress on the molecular biology of Mycoplasma suis[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):81-83.(in Chinese)
[8] 李松月,刘明明,贾立军.猪附红细胞体基因与蛋白研究进展[J].延边大学农学学报,2015,37(2):177-182. LI S Y,LIU M M,JIA L J.Recent advances of gene and protein for Eperythrozoon suis[J].Journal of Agricultural Science Yanbian University,2015,37(2):177-182.(in Chinese)
[9] HOELZLE K,DOSER S,RITZMANN M,et al.Vaccination with the Mycoplasma suis recombinant adhesion protein MSG1 elicits a strong immune response but fails to induce protection in pigs[J].Vaccine,2009,27(39):5376-5382.
[10] 薛书江,张守发,李海峰,等.猪附红细胞体α-烯醇化酶基因的克隆及生物信息学分析[J].畜牧与兽医,2014,46(5):31-34. XUE S J,ZHANG S F,LI H F,et al.Cloning and bioinformatics analysis of Eperythrozoon suis α-enolase gene[J].Animal Husbandry & Veterinary Medicine,2014,46(5):31-34.(in Chinese)
[11] 伍生军.猪附红细胞体ENO基因重组蛋白的制备及免疫效果评价[D].延吉:延边大学,2019. WU S J.The preparation of recobinant protein of Mycoplasma suis eno gene and evaluation of immune effect[D].Yanji:Yanbian University,2019.(in Chinese)
[12] SCHREINER S A,SOKOLI A,FELDER K M,et al.The surface-lacalised α-enolase of Mycoplasma suis is an adhesion protein[J].Veterinary Microbiology,2012,156(1-2):88-95.
[13] HOELZLE L E,HOELZLE K,HELBLING M,et al.MSG1,a surface-localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes[J].Microbes and Infection,2007,9(4):466-474.
[14] 李玉平,毛洪顺,潘连德,等.猪附红细胞体人工感染小鼠的病理学试验[J].中国兽医杂志,2010,46(8):40-41. LI Y P,MAO H S,PAN L D,et al.Pathological test of mice artificially infected with Mycoplasma suis[J].Chinese Journal of Veterinary Medicine,2010,46(8):40-41.(in Chinese)
[15] 杨朋艳,梁晚枫,张守发,等.猪附红细胞体感染BALB/c小鼠试验研究[J].中国农学通报,2012,28(23):69-72. YANG P Y,LIANG W F,ZHANG S F,et al.The infection test of Mycoplasma suis in BALB/c mice[J].Chinese Agricultural Science Bulletin,2012,28(23):69-72.(in Chinese)
[16] 余飞.猪附红细胞体小鼠感染模型的建立及血液学指标的检测[D].上海:上海交通大学,2008. YU F.Establishment of mice model with Eperythrozoon suis infection and detection of hematology changes[D].Shanghai:Shanghai Jiaotong University,2008.(in Chinese)
[17] 冯会权,徐昆,宋立先,等.猪附红细胞体感染BALB/c小鼠模型的建立及相关指标检测[J].畜牧与兽医,2011,43(1):63-65. FENG H Q,XU K,SONG L X,et al.Establishment of BALB/c mice model infected with Eperythrozoon suis and detection of related indexes[J].Animal Husbandry & Veterinary Medicine,2011,43(1):63-65.(in Chinese)
[18] 倪婷婷,伍生军,梁晚枫,等.猪附红细胞体小鼠感染模型建立条件的优化[J].中国畜牧兽医,2017,44(9):2761-2766. NI T T,WU S J,LIANG W F,et al.Optimization of establishment conditions of mouse model of Mycoplasma suis[J].China Animal Husbandry & Veterinary Medicine,2017,44(9):2761-2766.(in Chinese)
[19] 解海东.猪支原体肺炎灭活疫苗水性及水包油型佐剂研制及初步免疫效果评价[D].太谷:山西农业大学,2014. XIE H D.Study on development and immune enhancement effect of different aqueous and oil-in-water adjuvants to the Mycoplasma hyopneumoniae inactivated vaccine[D].Taigu:Shanxi Agricultural University,2014.(in Chinese)
[20] 董璐.猪支原体肺炎灭活疫苗及活疫苗佐剂研究[D].太谷:山西农业大学,2013. DONG L.Study on adjuvants of Mycoplasma hyopneumoniae inactivated vaccine and live vaccine[D].Taigu:Shanxi Agricultural University,2014.(in Chinese)
[21] BRADLEY L M,YOSHIMOTO K,SWAIN S L.The cytokines IL-4,IFN-γ,and IL-12 regulate the development of subsets of memory effector helper T cells in vitro[J].Journal of Immunology,1995,155(4):1713-1724.
[22] 李婷,包细明,鲜思美,等.羊IL-2和OrfV-F1L基因疫苗联合免疫小鼠的免疫应答研究[J].中国兽医科学,2015,3(6):124-126. LI T,BAO X M,XIAN S M,et al.Study on immune response in mice inoculated jointly with IL-2 gene vaccine and OrfV-F1L gene vaccine[J].Chinese Veterinary Science,2015,3(6):124-126.(in Chinese)
[23] 杨学琴,陈钰婷,李丽梅,等.不同刺激剂对人外周血淋巴细胞亚群增殖的影响[J].中国实验血液学杂志,2018,26(6):1765-1771. YANG X Q,CHEN Y T,LI L M,et al.Effects of different stimulators on proliferation of peripheral blood lymphocyte subsets[J].Journal of Experimental Hematology,2018,26(6):1765-1771.(in Chinese)
[24] 赵长祺,赵耀红,于海龙,等.木犀草素对小鼠T淋巴细胞体外增殖及凋亡的作用[J].中国实验方剂学杂志,2018,24(22):133-137. ZHAO C Q,ZHAO Y H,YU H L,et al.Effects of luteolin on proliferation and apoptosis of mouse T cells in vitro[J].Chinese Journal of Experimental Traditional Medical Formulae,2018,24(22):133-137.(in Chinese)