Cloning And Eukaryotic Expression Of PTN Gene In Antler Of Sika Deer

PTN(pleiotrophin)is a growth factor discovered and purified from bovine uterus in 1989 and has a high affinity with heparin.With the continuous development of the research,it was found that this factor is widely distributed and has multiple biological activities.Therefore,it was later renamed Pleiotropic Growth Factor.PTN is a 168 amino acid protein containing a signal peptide consisting of 32 amino acid residues and is highly conserved among species.Existing studies have shown that PTN also plays an important role in cancer cell proliferation.The antler regeneration and the magical proliferation speed are very rare in nature and therefore have become a hot topic of scientific research.Some scholars believe that the velvet antler horn regeneration mechanism is an ideal model for research on regenerative biology,and research on the pilose antler’s angle of proliferation mechanism may also become New breakthroughs in cancer treatment.According to the gene sequence of the homologous species in Genbank,homologous primers with digestion sites for PTN gene were designed.The cDNA sequence of PTN gene was obtained successfully using RT-PCR and molecular cloning techniques.The nucleotide sequence and amino acid sequence of the Sika deer pilose antler were compared.The nucleotide sequence of the PTN gene was 750 bp and encoded a total of 168 amino acids.Compared with the Blast function in NCBI,the results showed that the PTN gene of sika deer had higher homology with white-tailed deer,cattle and sheep,and the nucleotide sequences were 98%,97%,and 96%,which proved that the gene was relatively conservative in evolution and accorded with the characteristics of functional genes.The use of MEGA5.0 to construct a phylogenetic tree shows that sika deer are closely related to the evolution of white-tailed deer,cattle,sheep and other cloven-hoofed animals,and has a distant relationship with humans,Przewalski horses and North African fruit bats.This evolutionary feature is also consistent with classical taxonomy.The perspective reflects the reliability of the results of this study.Real-time PCR was used to detect the relative expression of PTN gene in antler tip tissue at different stages,the results showed that the medium-term expression level was higher than those of the previous and late periods.After activation of pMD-18T-PTN bacteriophage solution,the plasmid was extracted,and after double digestion,it was ligated with the eukaryotic expression vector pEGFP-C1 which was also digested with double enzyme,and the recombinant plasmid was transformed into DH5a cells and identification by PCR and double enzyme digestion.The identifications of PCR and double enzyme digestion showed that the pEGFP-Cl-PTN eukaryotic expression vector was successfully constructed.It was transiently transfected into resuscitate cultures and passaged twice in well-developed 293T cells.The green fluorescence was observed under a fluorescence microscope to prove that the transfection was successful.RNA was extracted from both experimental and control groups simultaneously.Real-time PCR showed that the expression level of PTN gene in the experimental group was about 16 times that of the control group,indicating that the pEGFP-C1-PTN eukaryotic expression vector was expressed successful in 293T cells.Real-time PCR detection revealed that when the PTN gene was overexpressed in 293T cells,the expression levels of HOXA5,MMP3,and MMP10 mRNA were significantly decreased,and EGF gene mRNA expression was significantly upregulated.