Construction Of Recombinant Porcine Sox2 And Nanog Lentiviral Vector

In this experiment, about 30-day-old fetal porcine were used for materials, and according to the published sequences of porcine Sox2, Nanog genes. A pair of specified primers containing the restriction sites were designed and synthesized. The BamHⅠand HindⅢrestriction sites were imported into primers of Sox2 and the XhoⅠand HindⅢrestriction sites also were imported into primers of Nanog. Total RNA was extracted from the fetal porcine genital ridge, and fragments of Sox2, Nanog gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR), digested with restriction endonuclease, and then connected with the retroviral vector pLEGFP-N1 and constructed recombinant plasmid PLEGFP-Sox2, PLEGFP-Nanog, PCR amplification of EGFP-Sox2, EGFP-Nanog and the transformation of the lentiviral vector pLentiLox3.7, Construction of recombinant plasmid PLL-Sox2, PLL-Nanog was transfected 293 cells by liposome-mediated the recombinant plasmid PLL-Sox2, PLL-Nanog. And then the Sox2, Nanog fusion of green fluorescent protein in 293 cells were observed after 24 hours.The results are as follows:(1) The sequence of porcine Sox2 and Nanog genes were cloned correctly. By RT-PCR reaction, about 957bp nucleotide sequence of the porcine Sox2 and 913bp nucleotide sequence of porcine Nanog were amplified. Correct sequence of porcine Sox2 and Nanog were connected to the T vector, Results showed that five base pairs of porcine Sox2 nucleotide sequence had difference compared with the wild boar by sequencing of Positive bacteria: the 224th base G→A, the 447th base C→T , the 511st base T→G, the 594th base C→A, the 712nd base A→G, the homology of it was 99.5%. The corresponding five amino acid had changed as the result of Codon degeneracy: the 75th Arg→Lys, the 149th site Arg→trp, 171 site Lys→Gly. Porcine Nanog the 913 bp nucleotide sequence also has 7 nucleotide base pairs have the difference from the wild boar : the 124th base T→G, the 258th base G→C, the 540th base C→T, the 602nd base G→A, the 610th base A→T, the 612nd base C→A, homology was 99.5%. The corresponding five amino acid changes: The 42nd site Ser→Ala, the 86th site Glu→Asn, the 201st site Ser→Thr, the 204th site Thr→Ser.(2) The recombinant retroviral expression vector plasmid PLEGFP-Sox2 and PLEGFP-Nanog was Successfully constructed. The correct sequence of the Sox2, Nanog were connected to pLEGFP-N1 vector and then constructed to the PLEGFP-Sox2, PLEGFP-Nanog. the results by PCR identification of 1% agarose gel electrophoresis showed that about the single band were 900 and 7 000 bp. The fragment size of Sox2 gene was about 957bp, the fragment size of Nanog was 913 bp, with the expected fragment consistent with the result. Recombinant plasmid were constructed and two bands were obtained, the size of them were about 900bp and 7 000bp, pLEGFP-N1 fragment size was 6891bp, were as the same as the RT-PCR product and the size of pLEGFP-N1 plasmid line. This will further comfirmed that the retroviral expression vector PLEGFP-Sox2, PLEGFP-Nanog were constructed successfully.(3) The recombinant lentiviral expression vector plasmid PLL-Sox2 and PLL-Nanog was Successfully constructed. Recombinant plasmid of PLL-Sox2, PLL-Nanog transfected 293 cells by using lipiosome-mediated vector, green fluorescence was observed 24 hours later. Under fluorescence microscope, 293 cells which have good shape can be seen, and 293 cells that transfected into PLL-Sox2, PLL-Nanog plasmid expressed EGFP, The resulet showed that the recombinant lentiviral expression vector plasmid PLL-Sox2 and PLL-Nanog were Successfully constructed. Lentiviral vector plasmid expressed Sox2, Nanog and EGFP. They can be transfected 293 cells.(4) The success of the reconstruction vector. Through the liposome-mediated transfection, The recombinant plasmid 293 cells was observed under fluorescence microscope. Lentiviral vector-mediated green fluorescent protein was expressed. The nuclear expression was in line with green fluorescent fusion protein designed, and this confirmed the vectors were reconstructed corrcetly.