The Cloning of St3gal Ⅰ Gene of SPF Chicken Embryo and Its Expression in MDCK Cells

doi:10.7668/hbnxb.2016.02.009

Abstract

Abstract: In order to improve the abundance of avian influenza virus(AIV) receptor on the surface of MDCK cells, 12-day-old SPF chicken embryos was as material and St3gal Ⅰ gene was cloned by RT-PCR.Next, the target fragment was inserted to the pMD18-T vector.Subsequently, the pMD18-T-St3gal Ⅰ and pCI-neo plasmids were digested and connected.Thus, the eukaryotic expression vector containing St3gal Ⅰ gene was constructed.Afterwards, the pCI-neo-St3gal Ⅰ plasmid was purified and transfected into MDCK cell by liposome-mediated method.The transgenic cell lines were screened by G418.The cell strains expressing St3gal Ⅰ gene stably were initially obtained by cell cloning culture and PCR assay.The results showed that St3gal Ⅰ gene from chicken embryo was successfully inserted into pCI-neo plasmid, leading to the construction of the eukaryotic expression vector pCI-neo-St3gal Ⅰ.In the G418 selection pressure, MDCK cells stably transfected target gene were initially obtained.The resistant cells were diluted, and 30 selected monoclonal resistant cell lines were selected.With genomic DNA and total RNA of transfected cells as a template, PCR analysis showed that the target gene had been integrated into the genome of MDCK cells and stably expressed.

Key words: Sialic acid, St3gal Ⅰ gene, MDCK, Transfection, G418, PCR

CLC Number:

LU Yujian, WU Xinming, GUAN Yu, ZHANG Songlin, LI Shufang, HAN Wenyu, SHEN Zhiqiang. The Cloning of St3gal Ⅰ Gene of SPF Chicken Embryo and Its Expression in MDCK Cells[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(2): 45-51. doi: 10.7668/hbnxb.2016.02.009.

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