Construction Of CDNA Library And CCL2 Gene Cloning And Expression In Erlunchun Horse

Erlunchun horse is one of China's important endangered species, the existing number of very small, less than 1,000, the cDNA library Construction of Erlunchun horse is of great significance for its protection of genetic resources ,and is very important for the reaseach of gene function .The study shows that the Erlunchun horse ear tissure cDNA library has been constructed successfully,the monocyte chemoattractant protein (MCP-1, macrophage chemotactic protein) gene, CCL2, has been cloned successfully, the Prokaryotic and eukaryotic expression vectors for CCL2 has been constructed smoothly , and the expression and subcellular localization of CCL2 has been implemented on the Mongolia horse fibroblast cells cultured in vitro.In this study,the total RNA had been extracted from the ear tissure of Erlunchun horse,and then the ear tissure cDNA library of Erlunchun horse had been constructed using SMARTTM technique.The identification result shows that the titer of unamplified cDNA library is 3.09×106pfu·mL-1,the rate of recombinant is above 93.75%, and the titer of amplified library is 1.4×1010pfu·mL-1,the average size of the fragments is 1.1 kb.This study is not only important for the structure and function research on horse genes,more importantly, is of great significance for its protection of genetic resources for Erlunchun horse.This study successfully established the target cell lines of Mongolia horse through direct adherent culture metheods,and the biological characteristics of detection had been done.The results shows that the quality of the cell line met the quality requirements of the ATCC (American Type Culture Collection). It indicate that the cells were well target cells for gene expression. In this study ,pGEX-4T-1-CCL2,the prokaryotic expression vector,had been constructed successfully , under the different final concentration gradient of IPTG, the SDS-PAGE results ,whitch was used to detect the pGEX-4T-1-CCL2 fusion protein, showed that the experimental group of about 36 kD, but all the differences between the experimental group was not obvious.All results means that the CCL2 gene had been expressed in BL21 successfully, but the further experiments should be done to optimize the conditions for induction.In this study,using the Erlunchun horse cDNA library as a template ,the gene CCL2 had been cloned successfully.The full length of CCL2 gene coding region was inserted into eukaryotic expression vectors pEGFP-N3,pEYFP-N1 and pDsRed1-N1 mul-cloning sites between EcoR I and Sal I, and construct recombinant eukaryotic expression vector pEGFP-N3-CCL2, pEYFP-N1-CCL2 and pDsRed1-N1-CCL2 with GFP as reporter gene.We used lipofectin method to transfect the recombinant vectors of pEGFP-N3-CCL2, pEYFP-N1-CCL2 and pDsRed1-N1-CCL2 into Mongolian horse fibroblast cells cultured in vitro,CCL2 on gene expression, subcellular localization, the positive cell growth and proliferation and viability was studied.The results showed that in transfected 24, 48 and 72 h, the reorganization of the fusion protein transfer rate is between 11.1 percent to 36.3 percent. Recombinant fusion protein in Mongolia horse fibroblast cells nucleus and cytoplasm showed the dispersive distribution.Recombinant fusion protein for CCL2 was expressed successfully in the fibroblast cells acording the RT-PCR amplification.