Abstract:

BACKGROUND: Adenovirus vector faces many problems, such as immunological rejection and inflammatory reaction, as an adipose tissue engineering of genetically modified vector. These problems can be avoided when using lentivirus as vector, especially using lentivirus carrier containing insulin gene. 
OBJECTIVE: To construct containing human recombinant insulin and enhanced green fluorescent protein (EGFP) gene lentiviral vector pLenti6.3-insulin IRES-EGFP, and to make virus particles packaging.
METHODS: Polymerase chain reaction (PCR) method was used to obtain the target gene and BamHⅠ, AscⅠ two restriction sites were added, after T vector cloning, the gene was transformed into competent DH5α cells. The gene fragment and pLenti6.3-IRES-EGFP vector were transformed into competent DH5α cells by means of enzyme digestion. The lentiviral expression vector pLenti6.3-insulin-IRES-EGFP was obtained after screening followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and the virus titers were determined.
RESULTS AND CONCLUSION: A length of 347 bp with BamH Ⅰ and Asc Ⅰ target gene sequences was obtained by PCR. The pMD18-T vector was connected to the lentiviral vector pLenti6.3 IRES-EGFP, and the constructed lentiviral expression vector pLenti6.3-insulin-IRES-EGFP was corresponded to the expected. The lentiviral particles were successfully packaged.