猪繁殖与呼吸综合征弱毒活疫苗免疫猪脾脏转移因子的制备及其活性分析

doi:10.11843/j.issn.0366-6964.2018.07.016

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (7): 1467-1474.doi: 10.11843/j.issn.0366-6964.2018.07.016

猪繁殖与呼吸综合征弱毒活疫苗免疫猪脾脏转移因子的制备及其活性分析

郭海勇¹, 袁洪兴², 孙泰然³, 曹静¹, 宋勤叶^2*, 李潭清², 逯纪成³

1. 吉林师范大学生命科学学院, 四平 136000;
2. 河北农业大学动物医学院河北省兽用生物制品工程技术研究中心, 保定 071001;
3. 河北省保定市动物疫病预防控制中心, 保定 071000

收稿日期:2018-01-13 出版日期:2018-07-23 发布日期:2018-07-19
通讯作者: 宋勤叶,Tel:0312-7528342,E-mail:songqinye@126.com
作者简介:郭海勇(1978-),男,吉林榆树人,博士,副教授,主要从事兽医新型生物制品研究,E-mail:guohaiyong78@163.com;袁洪兴(1980-),男,河北邯郸人,硕士,主要从事家畜传染病与新型生物制品研究,E-mail:yuanhongxing@126.com。
基金资助:
河北省科技计划项目（17226613D-2；13226603D-1<2013-X>）；河北省生猪产业技术体系疫病控制专项

Preparation and Immunological Activities of Transfer Factor from the Spleen of Pig Immunized with Porcine Reproductive and Respiratory Syndrome Attenuated-live Virus Vaccine

GUO Hai-yong¹, YUAN Hong-xing², SUN Tai-ran³, CAO Jing¹, SONG Qin-ye^2*, LI Tan-qing², LU Ji-cheng³

1. School of Life Science, Jilin Normal University, Siping 136000, China;
2. Hebei Province Research Center of Veterinary Biologicals Engineering and Technology, College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China;
3. The Center for Animal Disease Control and Prevention, Baoding 071000, China

Received:2018-01-13 Online:2018-07-23 Published:2018-07-19

摘要/Abstract

摘要：

从猪繁殖与呼吸综合征（PRRS）弱毒活疫苗免疫猪脾制备转移因子（TF），检测其免疫活性，为开发新型免疫增强剂和有效防制PRRS提供科学依据。用PRRSV弱毒活疫苗免疫健康育成猪，无菌采集脾，通过透析法从脾提取TF。应用磺基水杨酸法、茚三酮反应和硝酸-钼酸铵试验等方法检测TF的主要成分，经高效液相色谱法测定TF中氨基酸种类及其含量；通过鸡红细胞吞噬试验和MTT法，检测TF对小鼠腹腔巨噬细胞吞噬功能和外周血淋巴细胞增殖活性的调节作用。进而将9头4周龄健康仔猪随机分为A、B和C组，每组3头。A组每头猪左、右侧耳后分别肌内注射1头份PRRSV疫苗和1 mL TF；B组每头猪注射1头份PRRSV疫苗与1 mL TF的混合液；C组每头猪注射1头份PRRSV疫苗与1 mL生理盐水混合液。免疫后20 d，通过MTT法检测每组猪外周血淋巴细胞（PBLC）的增殖活性。制备的TF内无蛋白质成分，含有核酸和18种氨基酸，每毫升TF含核酸21.5 μg、总氨基酸3.479 mg。TF能显著增强小鼠腹腔巨噬细胞对鸡红细胞的吞噬能力和PBLC增殖活性。当TF与PRRSV疫苗分开或混合注射给4周龄仔猪后，能够显著提高仔猪PBLC的增殖能力。从PRRS弱毒活疫苗免疫猪的脾制备了TF，其能显著提高巨噬细胞的吞噬功能和外周血淋巴细胞的增殖活性。

关键词: 猪繁殖与呼吸综合征病毒, 猪, 脾, 转移因子, 免疫活性

Abstract:

The objective of this study was to prepare transfer factor(TF) from the spleen of pig immunized with porcine reproductive and respiratory syndrome(PRRS) attenuated-live virus vaccine, then detect its immune activities and expect to provide a scientific basis for developing a new immunopotentiator and prevention and control of PRRS. TF was extracted from spleen collected from healthy grown pigs which had been vaccinated with PRRS attenuated-live virus vaccine. The main components of TF were detected using sulfonyl salicylic acid, ninhydrin and ammonium molybdate-ammonium nitrate reactions, and kinds and contents of amino acids were measured by high performance liquid chromatography. The regulating effects on phagocytosis of peritoneal macrophage and lymphocytic proliferation of mice were checked by chicken erythrocyte phagocytosis test and MTT assay, respectively. Furthermore, nine piglets were divided into group A, B and C of 3 each. The piglets in the group A were injected intramuscularly with one dose of PRRS attenuated-live virus vaccine in left side opisthotic part and 1 mL of TF in the right side, respectively. Each one in the group B was injected intramuscularly the mixed liquor of one dose of PRRS attenuated-live virus vaccine with 1 mL of TF, and in the group C was did the mixed liquor of one dose of PRRS attenuated-live virus vaccine with 1 mL of normal saline. The proliferative activity of porcine peripheral blood lymphocyte(PBLC) from each group was measured by MTT on day 20 post-inoculation. There were nucleic acid and 18 kinds of amino acids in TF prepared in this study, containing 21.5 μg of nucleic acid and 3.479 mg total amino acids per mL of TF. Phagocytic ability of peritoneal macrophage to chicken erythrocytes and proliferative activity of PBLC from mice were enhanced significantly following stimulation of TF. Moreover, the proliferation capacity of porcine PBLC was also increased remarkably when the four-week-old piglets were injected with PRRS attenuated-live virus vaccine and TF simultaneously in different parts or their mixture. The transfer factor was prepared from the spleen of pig immunized with PRRS attenuated-live virus vaccine and it could increase significantly phagocytosis of macrophage and proliferative activity of PBLC.

Key words: PRRSV, pig, spleen, transfer factor, immunological activities

中图分类号:

S852.659.6

郭海勇, 袁洪兴, 孙泰然, 曹静, 宋勤叶, 李潭清, 逯纪成. 猪繁殖与呼吸综合征弱毒活疫苗免疫猪脾脏转移因子的制备及其活性分析[J]. 畜牧兽医学报, 2018, 49(7): 1467-1474.

GUO Hai-yong, YUAN Hong-xing, SUN Tai-ran, CAO Jing, SONG Qin-ye, LI Tan-qing, LU Ji-cheng. Preparation and Immunological Activities of Transfer Factor from the Spleen of Pig Immunized with Porcine Reproductive and Respiratory Syndrome Attenuated-live Virus Vaccine[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1467-1474.

参考文献

[1] 杨汉春, 周磊. 猪繁殖与呼吸综合征病毒的遗传变异与演化[J]. 生命科学, 2016, 28(3):325-336.
YANG H C, ZHOU L. Genetic variation and evolution of porcine reproductive and respiratory syndrome virus[J]. Chinese Bulletin of Life Sciences, 2016, 28(3):325-336. (in Chinese)
[2] RAHE M C, MURTAUGH M P. Mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus[J]. Viruses, 2017, 9(6):148.
[3] WANG G, LI L, YU Y, et al. Highly pathogenic porcine reproductive and respiratory syndrome virus infection and induction of apoptosis in bone marrow cells of infected piglets[J]. J Gen Virol, 2016, 97(6):1356-1361.
[4] HAN J, ZHOU L, GE X N, et al. Pathogenesis and control of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J]. Vet Microbiol, 2017, 209:30-47.
[5] NELSEN C J, MURTAUGH M P, FAABERG K S. Porcine reproductive and respiratory syndrome virus comparison:Divergent evolution on two continents[J]. J Virol, 1999, 73(1):270-280.
[6] 郭宝清, 陈章水, 刘文兴, 等. 从疑似PRRS流产胎儿分离PRRSV的研究[J]. 中国畜禽传染病, 1996, 18(2):1-5.
GUO B Q, CHEN Z S, LIU W X, et al. Isolation and identification of Porcine Reproductory and Respiratory Syndrome (PRRS) virus from aborted fetuses suspected of PRRS[J]. Chinese Journal of Animal and Poultry Infectious Diseases, 1996, 18(2):1-5. (in Chinese)
[7] TIAN K G, YU X L, ZHAO T Z, et al. Emergence of fatal PRRSV variants:Unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J]. PLoS One, 2007, 2(6):e526.
[8] CAO J, GRAUWET K, VERMEULEN B, et al. Suppression of NK cell-mediated cytotoxicity against PRRSV-infected porcine alveolar macrophages in vitro[J]. Vet Microbiol, 2013, 164(3-4):261-269.
[9] BAUMANN A, MATEU E, MURTAUGH M P, et al. Impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells[J]. Vet Res, 2013, 44:33.
[10] BAO D K, WANG R, QIAO S L, et al. Antibody-dependent enhancement of PRRSV infection down-modulates TNF-α and IFN-β transcription in macrophages[J]. Vet Immunol Immunopathol, 2013, 156(1-2):128-134.
[11] DONG J M, XU S G, WANG J, et al. Porcine reproductive and respiratory syndrome virus 3C protease cleaves the mitochondrial antiviral signalling complex to antagonize IFN-β expression[J]. J Gen Virol, 2015, 96(10):3049-3058.
[12] CHEN J, SHI X B, ZHANG X Z, et al. MicroRNA-373 facilitated the replication of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) by its negative regulation of type I interferon induction[J/OL]. J Virol, 2017, 91(3):e01311-16[2018-06-01]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5244336/.
[13] YOON I J, JOO H S, GOYAL S M, et al. A modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera[J]. J Vet Diagn Invest, 1994, 6(3):289-292.
[14] YOON K J, WU L L, ZIMMERMAN J J, et al. Antibody-dependent enhancement (ADE) of porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs[J]. Viral Immunol, 1996, 9(1):51-63.
[15] ROZZO S J, KIRKPATRICK C H. Purification of transfer factors[J]. Mol Immunol, 1992, 29(2):167-182.
[16] VIZA D, FUDENBERG H H, PALARETI A, et al. Transfer factor:an overlooked potential for the prevention and treatment of infectious diseases[J]. Folia Biol (Praha), 2013, 59(2):53-67.
[17] DUPONT B, BALLOW M, HANSEN J A, et al. Effect of transfer factor therapy on mixed lymphocyte culture reactivity[J]. Proc Natl Acad Sci U S A, 1974, 71(3):867-871.
[18] ROSENFELD S, DRESSLER D. Transfer factor:a subcellular component that transmits information for specific immune responses[J]. Proc Natl Acad Sci U S A, 1974, 71(6):2473-2477.
[19] 李士学, 侍俊超, 周微, 等. 特异性和非特异性猪脾转移因子的制备及其免疫活性比较[J]. 中国生物制品学杂志, 2011, 24(10):1190-1192.
LI S X, SHI J C, ZHOU W, et al. Preparation and immunological activity of specific and non-specific pig spleen transfer factor[J]. Chinese Journal of Biologicals, 2011, 24(10):1190-1192. (in Chinese)
[20] 叶兵兵, 李富桂. 鸡转移因子脂质体对小鼠巨噬细胞吞噬功能及肠绒毛长度的影响[J]. 中国畜牧兽医, 2014, 41(1):130-132.
YE B B, LI F G. Effects of chicken transfer factor liposome on macrophage phagocytosis and length of intestinal villus in mice[J]. China Animal Husbandry & Veterinary Medicine, 2014, 41(1):130-132. (in Chinese)
[21] ZHOU Y, BAI J, LI Y, et al. Suppression of immune responses in pigs by nonstructural protein 1 of porcine reproductive and respiratory syndrome virus[J]. Can J Vet Res, 2012, 76(4):255-260.
[22] HU H X, ZHANG X, ZHANG H S, et al. Porcine reproductive and respiratory syndrome virus inhibition of interferon-β transcription by IRF3-independent mechanisms in MARC-145 cells in early infection[J]. Vet Immunol Immunopathol, 2013, 156(1-2):135-140.
[23] ZHANG L, ZHOU L, GE X N, et al. The Chinese highly pathogenic porcine reproductive and respiratory syndrome virus infection suppresses Th17 cells response in vivo[J]. Vet Microbiol, 2016, 189:75-85.
[24] FABRE R A, PÉREZ T M, AGUILAR L D, et al. Transfer factors as immunotherapy and supplement of chemotherapy in experimental pulmonary tuberculosis[J]. Clin Exp Immunol, 2004, 136(2):215-223.
[25] 周建伟, 罗军敏, 孙万邦, 等. 甲型副伤寒特异性转移因子的制备和检定[J]. 西安交通大学学报:医学版, 2010, 31(5):637-639.
ZHOU J W, LUO J M, SUN W B, et al. Preparation and standardization of paratyphi a specific transfer factor[J]. Journal Xi'an Jiaotong University:Medical Sciences, 2010, 31(5):637-639. (in Chinese)
[26] XU Y P, ZOU W M, ZHAN X J, et al. Preparation and determination of immunological activities of anti-HBV egg yolk extraction[J]. Cell Mol Immunol, 2006, 3(1):67-71.
[27] 孔庆波, 陈进军, 常建军, 等. 猪犬脾TF的提取及理化性质和部分生物学特性测定[J]. 中国兽药杂志, 2007, 41(5):5-9.
KONG Q B, CHEN J J, CHANG J J, et al. The extraction of swine and canine spleen transfer factors and the determination of their physical and chemical properties and biological characteristics[J]. Chinese Journal of Veterinary Drug, 2007, 41(5):5-9. (in Chinese)
[28] FENG W H, TOMPKINS M B, XU J S, et al. Thymocyte and peripheral blood T lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus[J]. Virology, 2002, 302(2):363-372.
[29] PATEL D, NAN Y C, SHEN M Y, et al. Porcine reproductive and respiratory syndrome virus inhibits type I interferon signaling by blocking STAT1/STAT2 nuclear translocation[J]. J Virol, 2010, 84(21):11045-11055.
[30] BILODEAU R, ARCHAMBAULT D, VÉZINA S A, et al. Persistence of porcine reproductive and respiratory syndrome virus infection in a swine operation[J]. Can J Vet Res, 1994, 58(4):291-298.
[31] FONTANELLA E, MA Z X, ZHANG Y J, et al. An interferon inducing porcine reproductive and respiratory syndrome virus vaccine candidate elicits protection against challenge with the heterologous virulent type 2 strain VR-2385 in pigs[J]. Vaccine, 2017, 35(1):125-131.
[32] BATISTA L, PIJOAN C, DEE S, et al. Virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts[J]. Can J Vet Res, 2004, 68(4):267-273.

猪繁殖与呼吸综合征弱毒活疫苗免疫猪脾脏转移因子的制备及其活性分析

Preparation and Immunological Activities of Transfer Factor from the Spleen of Pig Immunized with Porcine Reproductive and Respiratory Syndrome Attenuated-live Virus Vaccine

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