东北白鹅CD8α基因的克隆及其胞外区基因的表达与抗血清的制备

doi:10.11843/j.issn.0366-6964.2015.04.023

Abstract

Abstract:

The aim of this study was to prepare antiserum against the goose CD8α protein.Specific primers for goose CD8α gene were designed according to public Mallard duck reference sequence (AF378373) in GenBank.Then Northeast White Goose CD8α gene was cloned by RT-PCR successfully.According to goose CD8α ORF sequence，specific primers were further designed and goose CD8α extracellular region was cloned.The recombinant goose CD8α extracellular region protein was expressed in prokaryotic expression system，pET-30a(+) and pET-28a(+) /Rosetta(DE3)pLysS.The recombinant protein was induced by IPTG and was purified by Ni-NTA column affinity chromatography.As immunogen，the purified recombinant protein stimulated the rabbit to produced anti-goose CD8α extracellular region serum.I-ELISA，Western blot analysis showed that antiserum could identify recombinant goose CD8α extracellular region protein and peripheral blood T lymphocytes respectively.Indirect immunofluorescence test confirmed that the purified antiserum could identify goose CD8α extracellular protein expressed by eukaryotic expression system specifically.The laser confocal scanning microscope had observed the fluorescence signal on the cell membrane.The above results show that antiserum can be used as detection reagent to detect the goose CD8⁺ T lymphocytes.

CLC Number:

S852.4

ZHANG Xue-lian，WEI Shuang-shi，LIU Xiao-mei，SHAO Jian-wei，ZHANG Shu-dong，LI Shan-shan，GAO Ming-chun，ZHANG Wen-long，XING Yu-gang，MA Bo，WANG Jun-wei. Molecular Cloning of Northeast White Goose CD8α Gene，Expression of Its Extracellular Region and Preparation of Corresponding Antiserum[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(4): 672-680.

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Molecular Cloning of Northeast White Goose CD8α Gene，Expression of Its Extracellular Region and Preparation of Corresponding Antiserum

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