The Construction And Foundational Identifycation Of Tau Gene Eukaryotic Expression Vector Of Guangxi Bama Mini-Pig

Tau protein has an important role in the formation and stability of cytoskeleton. Hyperphosphorylation of Tau protein will lead to neurofibrillary tangles, which is one of markers for Alzheimer’s disease. Thus, the objective of current study is to clone and construct the eukaryotic expression vector of Tau gene from Guangxi Bama mini-pig and to identify the function of recombinant plasmids. This study will make preparation for constructing the animal model of Alzheimer’s disease in Guangxi Bama mini-pig.The specific primers for Tau were designed according to predicted sequence released in GenBank. Total RNA was isolated and the coding sequence of Tau was amplified by reverse transcription (RT) PCR from brain tissue in Guangxi Bama mini-pig. There was a deletion of1134bp in Guangxi Bama mini-pig compared with the reference. The nested polymerase chain reaction (PCR) and5’rapid-amplification of cDNA ends (RACE) were employed to identify the deletions. The results showed that there was a deletion in the coding region (+344～+1389bp) and two alternative splicing (+191～+277bp,+1526～+1579bp),which led to the formations of four alternative variants. Those variants were released in GenBank and the accession numbers were KC473498, KC473499, KC473500, KC473501, respectively.Two fragments PDGF-β305and PDGF-β1501were amplified by PCR with primers, which were specific to human untranscription region of PDGF-B. The target fragments were inserted into the multi-clone sites of pEGFP-N1with a CMV promoter and pEGFP-1without a CMV promoter, respectively. The four recombinant plasmids were pPDGF-β305-EGFP-1, pPDGF-β305-EGFP-N1, pPDGF-β1501-EGFP-1, and pPDGF-β1501-EGFP-N1. The results of plasmids transfection in PC12cell indicated that PDGF-β305has a stronger effect in promoting the expression of EGFP from pEGFP-N1and pEGFP-1comparing with PDGF-β1501. Double promoters with PDGF-β305or PDGF-β1501and CMV have higher efficiency in promoting the gene expression comparing with single promoter with only PDGF-β305or PDGF-β1501. The pPDGF-β305-EGFP-1and pPDGF-β305-EGFP-Nl were transfected into PC12, PK15and C2C12cells. The results showed that the expression of EGFP was higher in PC12cell transfected with pPDGF-β305-EGFP-1comparing with pPDGF-β305-EGFP-1.Eukaryotic expression vector of Tau1215was constructed. The recombinant plasmids were pPDGF-β305-Taul215-EGFP-1and pPDGF-β305-Tau1215-EGFP-N1. The results of transfection of two plasmids in PC12were indicated that the expression of EGFP in pPDGF-β305-Tau1215-EGFP-N1was higher than that of in pPDGF-β305-Tau1215-EGFP-1. There were obvious changes in cellular morphology treated with pPDGF-β305-Taul215-EGFP-N1. Transgenic mouse with pPDGF-β305-Tau1215-EGFP-N1was conducted using the method of directly injection in ovary and testis. Rate of transgenic mice identified by PCR was13.9%in F1generation.In conclusions, Tau gene of Gauangxi Bama mini-pig was cloned and the two eukaryotic expression vectors were constructed. Those recombinant plasmids were expressed in cells. The study will make preparation for the construction of Alzheimer’s disease animal model in Guangxi Bama mini-pigs...