Cloning Of CD4 And CD8 Genes In Yanbian White Goose And Establishment Of SYBR Green ? Real-time PCR Method

CD4 and CD8 molecules are antigen receptors on the surface of T lymphocytes and belong to leukocyte differentiation antigens,which are involved in T cell activation and antigen presentation.The CD4 and CD8 molecules bind to MHC? and MHC I type molecules,respectively,and ensure the strong connection between T cells and the target cells.It is an important immune process in animal organisms.The cellular immunity level of animals can be observed by cloning and detecting CD4 and CD8 molecules.The experiment will be divided into two parts.The first chapter completes the cloning of CD4 and CD8 genes in Yanbian white goose and conducts preliminary bioinformatics analysis.In the second chapter,the cloned Yanbian white goose CD4 and CD8 genes were used as templates to establish the SYBR Green I real-time PCR method for CD4 and CD8 genes.First of all,Two pairs of primers for CD4 and CD8 genes were designed and amplified according to the two pairs of genomic sequences of CD4(GenBank:JQ272858)and CD8(GenBank:JQ272859)published by GenBank,respectively,in the blood of Yanbian white goose and the seven-day-old goose of Yanbian white goose.T lymphocytes were isolated from the thymus and the intracellular mRNA was extracted.The extracted mRNA was reverse transcribed,and then the designed primers were used to clone the CD4 and CD8 genes in the obtained cDNA,and sent to the sequencing company for identification and sequencing.Preliminary amino acid sequence analysis,homology analysis,and prediction of protein tertiary structure were performed.The results showed that the CD4 molecule of Yanbian white goose with a fragment size of 1443 bp and the CD8 molecule of Yanbian white goose with a fragment size of 711 bp were successfully cloned in this experiment.The tertiary structure prediction of the protein showed that the 480 amino acids encoded by the CD4 protein of Yanbian White Goose and the 236 amino acids encoded by Yanbian White Goose form a more complex high-level structure.Second,based on the principle of establishing an efficient,rapid and sensitive PCR method,the SYBR Green I real-time PCR method was selected in this experiment.The SYBR Green I real-time PCR method was selected for this experiment.SYBR Green I real-time PCR is performed using SYBR Green I fluorescent dye,and an excess of SYBR Green I fluorescent dye is incorporated into an optimized PCR system to bind the dye to the DNA duplex that requires specific amplification,The specific primers for the screening of CD4 and CD8 genes in the Northeast White Goose were designed to clone the fragments in the genome.The CD4 and CD8 genes that have been sequenced positive were used as templates for genomic cloning,and the cloned small fragments were ligated into pMD-18T.The carrier serves as a positive standard.Finally,a SYBR Green I real-time PCR method for quantitative detection of CD4 and CD8 genes was established.The results show that the linear relationship of this method is good,the correlation coefficient is only 0.999;the sensitivity is strong,which is 100 times the sensitivity of general PCR;the repeatability is strong,the difference between groups is small,and the coefficient of variation is less than 2.1t has the characteristics of high efficiency,rapidity and high sensitivity,and is a good method for detecting the number of CD4 and CD8 genes in goose.In summary,the CD4 and CD8 genes of Yanbian White Goose were successfully cloned in this experiment,and the bioinformatics analysis was successfully carried out.A highly efficient,rapid and sensitive SYBR GrenI real-time PCR method was successfully established.This study laid the foundation for the further production of monoclonal antibodies to CD4 and CD8 molecules of Yanbian White Goose and the subsequent detection of the expression of CD4 and CD8 molecules in vivo.It also provided a reference for the study of cloning and detection methods of other immune molecules.