[Construction of a recombinant Pim3-expressing plasmid and expression and activity thereof: experiment with rats].
OBJECTIVE
To construct a plasmid expressing green fluorescent protein (GFP) and gene of Pim-3, a member of the serine/threonine kinase family, and to investigate the in vivo expression of the construct and its effect on cell apoptosis.


METHODS
Pim-3 gene was cloned from myocardium tissues of Wistar rat by RT-PCR and subcloned into GFP-expressing plasmid vector pEGFP-N2 by restriction enzyme. The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified through enzyme digestion and gene sequencing. Thirty-two Wistar rats were randomly divided into 4 equal groups: Group A, as control group; Group B, injected intravenously with Ringer's solution; Group C, injected with blank vector, and Group D, injected with the recombinant plasmid pEGFP-N2/Pim-3. One day later, endotoxin/D-galactoamine (D-GalN) was intraperitoneally injected. 24 hours later the rats were killed. Fluorescence microscopy was used to observe the expression of the reporter gene GFP in the liver tissues. RT-PCR was used to detect the Pim-3 mRNA expression. The hepatic apoptosis was detected by TUNEL assay. The activity of caspase-3 was detected.


RESULTS
A 998 bp target cDNA fragment with restriction enzyme sites was amplified and inserted into the multiple clone site of pEGFP-N2 successfully. High expression levels of the target gene Pim-3 and reporter gene GFP were achieved in the rat liver after transfer of the recombinant plasmid. The relative Pim-3 expression level of Group D was 0.49 +/- 0.15, significantly higher than those of Groups A, B, and C (0.06 +/- 0.02, 0, and 0 respectively, all P < 0.01). The apoptotic index of Group D was (4.9 +/- 1.2)%, significantly lower than those of Groups B and C [(72.5 +/- 6.1)% and (69.8 +/- 5.7)% respectively, both P < 0.01]; however, not significantly different from that of Group A [(3.1 +/- 0.7)%]. The activity of caspase-3 of Group D was (76 +/- 27) pmol.min(-1).mg(-1), significantly lower than those of Groups B and C [(147 +/- 55) and (142 +/- 50) pmol.min(-1).mg(-1), respectively, both P < 0.01]; however, not significantly different from that of Group A (60 +/- 15).


CONCLUSIONS
The recombinant plasmid pEGFP-N2/Pim-3 can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis.
Liu
Liang-Ming
LM
Department of Gastroenterology, the Second Affiliated Hospital, Nanchang University, Jiangxi Province Key Laboratory of Molecular Medicine, Nanchang 330006, China.
Deng
Huan
H
Zhang
Ji-Xiang
JX
Luo
Jie
J
Yin
Dong
D
Guo
Hong-Xing
HX
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D004731:Endotoxins / Q000494:pharmacology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004731:Endotoxins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C113398:Pim3 protein, rat; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3