China Animal Husbandry and Veterinary Medicine ›› 2020, Vol. 47 ›› Issue (10): 3088-3094.doi: 10.16431/j.cnki.1671-7236.2020.10.006

• Biotechnology • Previous Articles     Next Articles

Construction and Identification of Lentiviral Overexpression Plasmid of Mouse Zbtb38 Gene

LI Jie1, CHEN Jing1, YANG Fan1, LI Jun1, CAI Yafei2   

  1. 1. Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, College of Life Sciences, Anhui Normal University, Wuhu 241000, China;
    2. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
  • Revised:2020-05-27 Online:2020-10-20 Published:2020-10-17

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Abstract: This study was conducted to construct zinc finger and BTB domain containing 38 (Zbtb38) gene lentiviral overexpression plasmid vector.Firstly,the CDS sequence of mouse Zbtb38 gene was amplified by overlap extension PCR method using mouse spinal cord tissue,and the Zbtb38 CDS sequence front amplification primer 1 and the latter amplification primer 2 were designed respectively.Using the amplification products of the above two primers as template,primer 3 was designed and subjected to fusion PCR amplification,thereby obtaining the full CDS sequence of Zbtb38.Then,the obtained Zbtb38 gene CDS full sequence was ligated to the plasmid pGM-T,and the recombinant plasmid was transferred into E.coli DH5α competent cells,the positive clones were identified and verified by direct PCR and sequencing.Finally,the Zbtb38 CDS sequence was ligated into the lentiviral vector Plvx-puro by XhoⅠ and BamHⅠ double digestion to obtain the shuttle plasmid Plvx-puro-Zbtb38,which was co-transfected with lentiviral packaging plasmids to produce lentiviral particles in 293T cells.The results showed that the primer 1 amplification product contained the first 1-1 794 bp of the Zbtb38 CDS sequence,and the actual sequencing length of the amplified product was 1 772 bp.The primer 2 amplification product contained the 1 762-3 594 bp of the entire sequence of Zbtb38 CDS,and the actual sequencing length of the amplified product was 1 818 bp,indicating that the fragmented amplification target fragment was successfully obtained.The expected fusion products amplified by primer 3 and the PCR amplification products of the colonies both contained the complete sequence of Zbtb38 CDS (3 594 bp),which was compared with the CDS sequence of Zbtb38 gene in NCBI database,and the coincidence rate was 99.99%,indicating that the Zbtb38 CDS sequence was successfully amplified.The Real-time PCR results showed that the virus titer reached 3.25×108 Tu/mL,which met the experimental animal injection standard requirement,indicating that the lentiviral vector was successfully constructed.In summary,the mouse Zbtb38 gene lentiviral overexpression plasmid vector was successfully constructed in this experiment.

Key words: Zbtb38 gene; overlap PCR; pGM-T plasmid; lentiviral vector

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