Construction and expression of attenuated salmonella typhimurium carrying human adiponectin gene eukaryotic expression vector
AIM: To construct and express an attenuated Salmonella typhimurium strain contraining human adiponectin gene eukaryotic expression vector, in order to lay the foundation for the genetic therapy of nonalcoholic fatty liver disease in future. METHODS: Total mRNA was extracted from human fat tissue and the adiponectin cDNA was obtained by RT-PCR, and then cloned into eukaryotic expression vector pEGFP-N1. The recombinant vector was identified, and transformed into attenuated Salmonella typhimurium strain SL7207, and the recombinant strain SL7207/pEGFP-NI-AdipoQ was screened by green fluorescent microscope and Western Blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes was found to be 744 bp, and had been inserted into eukaryotic expression vector, but as compared with gene reported by GenBank, 0. 1% of the gene mutation and O. 4% of amino acid residues change, respectively: 654 bp: A→T, amino acids: Glu→Asp. SDS-PAGE demonstrated that pEGFP-N1-AdipoQ was expressed in SL7207 strain, and the relatived molecular mass of the GFP-AdipoQ fusion protein was about 55×10~3, while GFP protein expressed by pE(;FP-NI was about 27×10~3. The Western Blot analysis indicated that the EGFP-AdipoQ fusion protein could be recognized by anti-AdipoQ antibody, suggesting that this fusion protein had good biological activity. CONCLUSION: The recombinant attenuated Salmonella typhimurium strain contraining plasmid pEGFP-N1- AdipoQ can be successfully constructed and expressed, and can be a basic foundation for the genetic therapy of NFLD in future with adiponectin..
NETL
NSTL
万方数据
维普
