鸡斑联蛋白基因的慢病毒干扰载体的构建及筛选

doi:10.11843/j.issn.0366-6964.2013.08.023

Abstract

Abstract:

The aim of this study was to construct 4 Lenti-virus Interference Vectors for chicken zyxin gene, determine their titers, and filter the effective interference target, and lay the foundation for studying of anti-coccidial mechanism of chicken zyxin gene. The experiment was performed by extracting RNA from chicken thymus, then synthesizing the cDNA with reverse transcription, amplifying coding regions of zyxin, and cloning it into eukaryotic expression vector Pex-6 at last. The shRNA sequences for zyxin were designed, and were inserted into Lv3 lenti-virus vectors. Lv3-shRNA vectors and packaging plasmids which include pGag/Pol, pRev and Pvsv-G were cotransfected into 293T cells to package virus. Dilute virus liquid 10 times the 6 gradients, and transfect them into 293T cells respectively to determine the titer. Transfect 4 recombinant lenti-virus interference vectors and eukaryotic expression vectors Pex-6 into 293T cells, and filter the effective interference target by RT-RCR and Western-blot. The sequence identification results showed that the 4 constructed zyxin recombinant lenti-virus interference vectors including CO1, CO2, CO3, CO4 and a negative control plasmid were correctly constructed. By way of transfecting them into 293T cells and concentration, the titer reached 1×10⁸ TU·mL^－1 which showed that they were suit for being transfected into the target cells. The filter result showed that CO3 had the most significant interference effect on zyxin expression in 293T cells. Analysis of the data suggests that the zyxin recombinant lenti-virus interference vectors were constructed and filtered the effective interference target successfully, which laid the foundation for further research on zyxin gene function.

CLC Number:

S813.3

DAI Guo-jun, SUN Da-hui, LIN Yu-xin, SUN Ming-ming, WANG Xiang, WANG Jin-yu, ZHANG Gen-xi, XIE Kai-zhou, SHI Hui-qiang, SHI Miao-miao. Construction and Selection of Chicken Zyxin Recombinant Lenti-virus Interference Vectors[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(8): 1330-1336.

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References

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Construction and Selection of Chicken Zyxin Recombinant Lenti-virus Interference Vectors

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