[1]王美淇?,李远超?,李文佳,等.延边白鹅CD4与CD8基因SYBR Green I 荧光定量PCR方法的建立与应用[J].中国预防兽医学报,2019,(12):1238-1243.[doi:0.3969/j.issn.1008-0589.201908015]
 WANG Mei-qi,LI Yuan-chao,LI Wen-jia,et al.Establishment and application of SYBR Green I real-time PCR for the detection of CD4 and CD8 genes in Yanbian white goose[J].Chinese journal of preventive veterinary medicine,2019,(12):1238-1243.[doi:0.3969/j.issn.1008-0589.201908015]
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延边白鹅CD4与CD8基因SYBR Green I 荧光定量PCR方法的建立与应用()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年12
页码:
1238-1243
栏目:
诊断和检测技术
出版日期:
2020-01-25

文章信息/Info

Title:
Establishment and application of SYBR Green I real-time PCR for the detection of CD4 and CD8 genes in Yanbian white goose
文章编号:
1008-0589(2019)12-1238-06
作者:
 

王美淇?李远超?李文佳陈 姗孟 媛刘晋宇高 旭*

Author(s):
 

WANG Mei-qi? LI Yuan-chao? LI Wen-jia CHEN Shan MENG Yuan LIU Jin-yu GAO Xu*

 (Department of Veterinary Medicine, Agriculture College of Yanbian University, Yanji 133000, China)
关键词:
延边白鹅CD4CD8SYBR Green I 荧光定量PCR
Keywords:
 Yanbian white goose  CD4  CD8  SYBR Green I real-time PCR
分类号:
S858.31
DOI:
0.3969/j.issn.1008-0589.201908015
文献标志码:
A
摘要:
为建立延边白鹅CD4与CD8基因在细胞免疫中的动态变化的检测方法,本研究根据CD4与CD8基因保守区分别设计两对特异性引物,在克隆目的基因的基础上,进行TA克隆和转化,构建的阳性质粒作为标准品,经反应条件优化建立了检测延边白鹅CD4与CD8基因的荧光定量PCR方法,并进行特异性、敏感性和重复性试验。结果显示,建立的SYBR Green I荧光定量PCR方法在质粒标准品稀释倍数为101~108倍(CD4:1.2×108拷贝/μL~1.2×101拷贝/μL;CD8:1.8×108拷贝/μL~1.8×101拷贝/μL)范围内具有良好的线性关系,相关系数均为0.999,与其它相关鹅细胞因子基因不发生交叉反应,敏感性均是普通PCR的100倍,组内和组间变异系数均小于2 %。初步应用本研究建立的方法检测鹅脾脏淋巴细胞CD4与CD8基因的转录水平,结果显示ConA可诱导目的基因转录水平升高,且显著高于PBS对照组(p<0.05)。本研究建立的SYBR Green I荧光定量PCR方法为进一步研究鹅细胞免疫水平和CD4、CD8生物学活性奠定了基础。
  
Abstract:
In order to establish one method for the quantification of CD4 and CD8 levels during the cell immune response in Yanbian white goose, two pairs of specific primers were designed in the conserved region of the target gene. After the PCR amplification, the obtained amplicons were cloned and transformed into E.coli, the resulting recombinant plasmid was used as the standard. The real-time quantitative PCR for the detection of CD4 and CD8 genes in Yanbian white goose was developed by optimizing the reaction conditions. And the sensitivity, specificity and reproducibility were evaluated. The results showed that the established SYBR Green I real-time PCR had a good linear relationship in the range of 101-108 copies/μL, the correlation coefficient was 0.999, and the method had no cross reaction with other goose cytokine genes. The sensitivity of this assay was 100 times more than that of ordinary PCR. The intra-asssay and inter-assay of coefficients are all less than 2%. The transcription levels of CD4 and CD8 in the goose lymphocytes stimulated by Concanavalin A (ConA) were detected by using this assay. The results showed that the transcription level of the target genes were increased significantly after ConA induction compared with that for PBS control group (p<0.05). Therefore, the SYBR Green I real-time PCR method established in this study laid the foundation for the further study of cell immune level and CD4, CD8 biological activity in goose.

参考文献/References:

[1]金伯泉. 细胞和分子免疫学[M]. 第二版. 北京:科学出版社,2001.
[2] Chan M M, Chen C L, Ager L L, et al. Identification of the avian homologues of mammalian CD4 and CD8 antigens [J]. J Immunol, 1988, 140(7): 2133-2138.
[3]Zhou Xiao-lu, Wu Shan-li, Zhou Hong-da, et al. Marek’s disease virus regulates the ubiquitylome of chicken CD4+ T Cells to promote Tumorigenesis [J]. Int J Mol Sci, 2019, 20: 2-16.
[4]Xing Xing-Jing, Ma Jun-jie, Tang Xiao-qian, et al. Characterizations of CD4-1, CD4-2 and CD8β T cell subpopulations in peripheral blood leucocytes, spleen and head kidney of Japanese flounder (Paralichthysolivaceus)[J]. Mol Immunol, 2017, 85: 155-165.
[5]Castilho J L, Turner M, Shepherd B E, et al. CD4/CD8 ratio and CD4 nadir predict mortality following non-communicable disease diagnosis in adults living with HIV [J]. AIDS Res Hum Retroviruses, 2019, 12: 741-752.
[6]Chen Shun, Zhou Qin, Cheng Bei-bei, et al. Age-related development and tissue distribution of T cell markers (CD4 and CD8a) in Chinese goose [J]. Immunobiology, 2015, 220(6):753-761.
[7]张伟,程蓓蓓,陈舜,等. 基于鹅T细胞表面CD4分子胞外区的双抗体夹心ELISA检测方法的建立[J]. 中国预防兽医学报,2016,38(9):729-733.
[8]张雪莲,魏双施,邵建伟,等. 东北白鹅CD4基因的克隆及其胞外区的表达与抗血清的制备[J]. 畜牧兽医学报,2014,45(4):639-646.
[9]Kothlow S, Mannes N K, Schaerer B, et al. Characterization of duck leucocytes by monoclonal antibodies [J]. Dev Comp Immunol, 2005, 29(8): 733-748.
[10]Yan Xiao-ling, Liu Fei, Chen Shun-yan, et al. Molecular cloning characterization and tissue expression of CD4 in Chinese goose [J]. Gene, 2013, 519(2): 298-304.
[11]李卓昕,温树波,孙文超,等. 猪圆环病毒3型SYBR Green Ⅰ实时荧光定量PCR方法的建立及应用[J]. 中国病原生物学杂志,2018,13(9):934-938.
[12]Shweta S, Indranil D. Development of SYBR Green I based real-time PCR assays for quantitative detection of rice tungro bacilliform virus and rice tungro spherical virus [J]. J Virol Methods, 2012, 181(1): 86-92.
(本文编辑:彭永刚;英文编辑:胡 哲)

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更新日期/Last Update: 2020-01-19