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  

Establishment and application of SYBR Green I real-time PCR for the detection of CD4 and CD8 genes in Yanbian white goose

作　　者：王美淇 李远超 李文佳 陈姗 孟媛 刘晋宇[1] 高旭[1] 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)

机构地区：[1]延边大学农学院动物医学系,吉林延吉133000

出　　处：《中国预防兽医学报》2019年第12期1238-1243,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金项目(31460661)。

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(CD4:1.2×10^8拷贝/μL^1.2×10^1拷贝/μL;CD8:1.8×10^8拷贝/μL^1.8×10^1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。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.

关 键 词：延边白鹅 CD4 CD8 SYBR Green I荧光定量PCR 

分 类 号：S858.31[农业科学—临床兽医学]