山羊Th1/Th2细胞因子实时荧光定量检测方法的建立
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摘要
目的在mRNA水平对山羊Th1/Th2型细胞因子进行定量分析,建立山羊Th1/Th2型细胞因子实时荧光定量检测方法。方法根据GenBank山羊Th1(IL-2和IFN-γ)和Th2(IL-4、IL-6和IL-10)细胞因子基因保守序列设计引物,以标准阳性质粒为模板分别进行荧光定量PCR反应的标准曲线、溶解曲线建立。结果山羊Th1/Th2细胞因子实时荧光定量检测方法Ct值与标准品呈良好的线性关系,R~2均大于0.985,所有稀释度标准品模板出现特异性熔解峰;利用N1蛋白缺失的重组山羊痘病毒(△N1L株)与山羊痘AV41株感染山羊外周血淋巴细胞进行IL-4和IFN-γmRNA表达水平检测,△N1L株与山羊痘AV41株均能能够刺激机体IL-4和IFN-γ细胞因子,但二者差异无统计学意义(t=3.333,P>0.5)。结论本研究建立山羊Th1/Th2细胞因子实时荧光定量检测,为山羊痘基因工程疫苗的研究奠定基础。
This study was to establish a real-time fluorescence quantitative PCR method for detection of goat's Th1/Th2 cytokines.The conserved region of goat's Th1/Th2 cytokines gene were amplified and cloned into pMD18-T vector.The plasmid DNA was used as template to optimize assay condition of developing a SYBR GreenⅠReal-time PCR for Th1/Th2 cytokines detection.The results showed that the Ct value of Th1/Th2 cytokines produced a good linear relationship, which R~2 were all greater than 0.985.All standards were specific narrow melting peak and reproducetire and specific.The established assays were successfully used to detect IL-4 and IFN-γmRNA expression levels in peripheral blood mononuclear cells(PBMCs) in sheep experimentally infected with recombinant goat poxstrain(△N1 L strain) and AV41 strain, both could stimulate Th1/Th2 cytokines with no difference in vivo. In this study, real-time fluorescence quantitative detection of goat Th1/Th2 cytokines was established, which laid the foundation for the research of goat pox genetic engineering vaccine.
This study was to establish a real-time fluorescence quantitative PCR method for detection of goat's Th1/Th2 cytokines.The conserved region of goat's Th1/Th2 cytokines gene were amplified and cloned into pMD18-T vector.The plasmid DNA was used as template to optimize assay condition of developing a SYBR GreenⅠReal-time PCR for Th1/Th2 cytokines detection.The results showed that the Ct value of Th1/Th2 cytokines produced a good linear relationship, which R~2 were all greater than 0.985.All standards were specific narrow melting peak and reproducetire and specific.The established assays were successfully used to detect IL-4 and IFN-γmRNA expression levels in peripheral blood mononuclear cells(PBMCs) in sheep experimentally infected with recombinant goat poxstrain(△N1 L strain) and AV41 strain, both could stimulate Th1/Th2 cytokines with no difference in vivo. In this study, real-time fluorescence quantitative detection of goat Th1/Th2 cytokines was established, which laid the foundation for the research of goat pox genetic engineering vaccine.
引文
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[12] 张贺楠, 赖汉漳, 齐岩,等. 鸡IFN-α和IFN-β及IFN-γ基因实时荧光定量RT-PCR检测方法的建立 [J].中国兽医科学, 2009, 39(02): 173-177.
[13] Chkhaidze I, Zirakishvili D, Shavshvishvili N, et al. Prognostic value of TH1/TH2 cytokines in infants with wheezing in a three year follow-up study [J]. Pneumonol Alergol Pol, 2016, 84(3): 144-150. DOI: 10.5603/PiAP.2016.0016
[14] George JA, Kim SB, Choi JY, et al. TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity [J]. Oncotarget, 2017, 8(62): 106050-106070. DOI: 10.18632/oncotarget.22525
[15] Zhang W, Pan Y, Gou P, et al. Effect of xanthohumol on Th1/Th2 balance in a breast cancer mouse model [J]. Oncol Rep, 2018, 39(1): 280-288. DOI: 10.3892/or.2017.6094
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[17] Barton SJ, Ngo S, Costello P, et al. DNA methylation of Th2 lineage determination genes at birth is associated with allergic outcomes in childhood [J]. Clin Exp Allergy, 2017,47(12): 1599-1608. DOI: 10.1111/cea.12988
[18] We Naugler, T Sakurai, S Kim, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production [J]. Science, 2007, 317(5834): 121-124. DOI: 10.1126/science.1140485
[19] De Waal Malefyt R, Abrams J, Bennett B, et al. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes [J]. J Exp Med, 1991, 174(5): 1209-1220. DOI: 10.1084/jem.174.5.1209
[2] Wongyanin P, Buranapraditkun S, Chokeshai-Usaha K, et al. Induction of inducible CD4+CD25+Foxp3+ regulatory T lymphocytes by porcine reproductive and respiratory syndrome virus (PRRSV) [J]. Vet Immunol Immunopathol, 2010, 133(2/4):170-182. DOI: 10.1016/j.vetimm.2009.07.012
[3] Abo-Aziza FAM, Hendawy SHM, Namaky AHE, et al. Th1/Th2 balance and humoral immune response to potential antigens as early diagnostic method of equine Strongylus nematode infection [J]. Vet World, 2017, 10(6): 679-687. DOI: 10.14202/vetworld.2017.679-687
[4] Li X, Huang L, Wang N, et al. Sulfur dioxide exposure enhances Th2 inflammatory responses via activating STAT6 pathway in asthmatic mice [J]. Toxicol Lett, 2017, 285: 43-50. DOI: 10.1016/j.toxlet.2017.12.020
[5] Nakajima R, Miyagaki T, Oka T, et al. IL-25 is involved in CTCL progression by establishing Th2-dominant microenvironment [J]. Br J Dermatol, 2017,178(6):1373-1382. DOI: 10.1111/bjd.16237
[6] Mitson-Salazar A, Prussin C. Pathogenic effector th2 cells in allergic eosinophilic inflammatory disease [J]. Front Med (Lausanne), 2017, 4: 165. DOI:10.3389/fmed.2017.00165
[7] Hemann M, Shen HG, Beach NM, et al. Expression of human CD46 has no effect on porcine circovirus type 2 infection and shedding in the experimental pig model [J]. Vet Res Commun, 2012, 36(3): 187-93. DOI: 10.1007/s11259-012-9524-z
[8] Ahmad SF, Zoheir KMA, Ansari MA, et al. Dysregulation of Th1, Th2, Th17, and T regulatory cell-related transcription factor signaling in children with autism [J]. Mol Neurobiol, 2017, 54(6): 4390-4400. DOI: 10.1007/s12035-016-9977-0
[9] 施开创, 李焕荣, 杨汉春,等.猪IL-4、IL-6和IL-10 SYBR Green Ⅰ real-time PCR检测方法的建立及应用 [J].中国预防兽医学报, 2010, 32(2): 102-107.
[10] 韩猛立, 蔡曰鹏, 黄新,等. 牛Th1/Th2型细胞因子实时荧光定量RT-PCR检测方法的建立及应用 [J].中国兽医学报, 2011, 31(04): 513-520.
[11] 张亮亮, 侯小强, 高玉伟,等.小鼠细胞因子实时定量PCR检测方法的建立及应用 [J].中国兽医学报, 2009, 29(12): 1552-1556.
[12] 张贺楠, 赖汉漳, 齐岩,等. 鸡IFN-α和IFN-β及IFN-γ基因实时荧光定量RT-PCR检测方法的建立 [J].中国兽医科学, 2009, 39(02): 173-177.
[13] Chkhaidze I, Zirakishvili D, Shavshvishvili N, et al. Prognostic value of TH1/TH2 cytokines in infants with wheezing in a three year follow-up study [J]. Pneumonol Alergol Pol, 2016, 84(3): 144-150. DOI: 10.5603/PiAP.2016.0016
[14] George JA, Kim SB, Choi JY, et al. TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity [J]. Oncotarget, 2017, 8(62): 106050-106070. DOI: 10.18632/oncotarget.22525
[15] Zhang W, Pan Y, Gou P, et al. Effect of xanthohumol on Th1/Th2 balance in a breast cancer mouse model [J]. Oncol Rep, 2018, 39(1): 280-288. DOI: 10.3892/or.2017.6094
[16] Kondo M, Takeshita T, Ishii N, et al. Sharing of the interleukin-2 (IL-2) receptor gamma chain between receptors for IL-2 and IL-4 [J]. Science, 1993, 262(5141): 1874. DOI: 10.1126/science.8266076
[17] Barton SJ, Ngo S, Costello P, et al. DNA methylation of Th2 lineage determination genes at birth is associated with allergic outcomes in childhood [J]. Clin Exp Allergy, 2017,47(12): 1599-1608. DOI: 10.1111/cea.12988
[18] We Naugler, T Sakurai, S Kim, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production [J]. Science, 2007, 317(5834): 121-124. DOI: 10.1126/science.1140485
[19] De Waal Malefyt R, Abrams J, Bennett B, et al. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes [J]. J Exp Med, 1991, 174(5): 1209-1220. DOI: 10.1084/jem.174.5.1209
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