Construction Of PGC-FU-survivin Lentivirus Expression Vector

Background and ObjectiveHepatitis B is a highly prevalent disease in China,which subsequently results in a high incidence of fulminant hepatitis B. Fulminant hepatitis is characterized by severe and extensive hepatocytes necrosis. Owing to the emergency of disease, limit of investigations and inefficient therapeutic strategy, fulminant hepatitis leads to a high morbidity. So far, there have been no effective treatments to fulminant hepatitis. Liver transplantation is recognized to be the key solution. However, high expenses, scanty source of donor liver and grave rejection reaction have diminished its prosperity. Comparing to other source of cells, primary hepatocytes which resemble the normal hepatocytes most, could be used for cell transplantation. However, the low transfection rate and passage difficulty demand a lentivirus-induced infection, through which virus could stably integrate into the host gene and passage the gene we need to next generations.Survivin is considered to be the strongest protein that regulates cell proliferation and apoptosis, and there may be certain relationships between survivin and hepatocyte proliferation. As a result, to construct a pGC-FU-survivin lentivirus plasmid and to study its further biological functions are of great value. Methods1. Mouse survivin gene was obtained from YAC-I cell line, a mouse lymphoma cell line, which highly expresses survivin protein, then the gene was linked to pcDNA3.1 (+) plasmid. The accuracy was determined by restriction endonuclease digestion and sequencing.2. Survivin gene was acquired from pcDNA3.1 (+)-survivin plasmid and then connected to linearized pGC-FU plasmid, a lentivirus vector, then the accuracy was determined by sequencing and Western Blotting. After package and purifying, high titer of virus particles were established for further study.3. pGC-FU-survivin plasmid was transfected to mouse primary hepatocytes.Results1. Restriction endonuclease digestion and sequencing confirmed that survivin obtained from YAC-I cell line was connected to pcDNA3.1 (+) plasmid correctly. Comparing to survivin gene nucleotides in Gene Bank, all the sequences coincided with the Bank sequences except an A to T mutation at 317th gene position, which attributed an aminoglutaminic acid to aminoisovaleric acid at 68th protein position, but didn't affect spacial configuration and biological functions of survivin protein, according to NCBI protein library.2. After transfecting pGC-FU-survivin plasmid to 293T cells, green fluorescence was obviously observed. A 43kD protein was gotten from Western Blotting; it was approximate the same molecular weight as the standard (46kD, the standard is a little bit bigger in molecular weight than survivin-GFP fusion protein). Packaging virus particles in 293T cells, after purifying, high titer (2×108TU/mL) of pGC-FU-survivin plasmid was received eventually.3. pGC-FU-survivin plasmid can be transfected into mouse hepatocytes succesfully; however, the transfection rate was still unsatisfied. ConclusionpGC-FU-survivin plasmid was constructed successfully with the assistance of pMD18-T vector and pcDNA3.1 (+) plasmid.