[Construction and identification of eukaryotic co-expression plasmid carrying FUS1 and recombinant human IL-12 genes and its effection on apoptosis of A549 cells].
OBJECTIVE
To construct a eukaryotic co-expression plasmid containing FUS1 and IL-12, and to investigate the influence of the recombinant plasmid on the cultured human lung cancer cell line A549.


METHODS
RT-PCR was applied to amplify FUS1 from MRC-5 cell, the cDNA fragment of IL-12 was derived from pORF-hIL-12 by PCR, FUS1 cDNA fragment and IL-12 cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-FUS1-IL-12. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequenceing and transfected into A549 cells. The expression of genes in pVITRO2-FUS1-IL-12 were identified by RT-PCR, ELISA and Western blot. The function of pVITRO2-FUS1-IL-12 inducing the apoptosis of lung cancer was identified by Hoechst33258 and flow cytometry.


RESULTS
The restriction endonuclease digestion and sequencing suggested that co-expression vector pVITRO2-FUS1-IL-12 was constructed successfully, the expression of FUS1 and IL-12 could be detected in A549 cells. The expressions of the two genes in pVITRO2 were not affected. The A549 cells transfected with pVITRO2-FUS1-IL-12 exhibited significant cell apoptosis.


CONCLUSIONS
The recombinant eukaryotic expression vector pVITRO2-FUS1-IL-12 was constructed and expressed in eukaryotic cell successfully, which would contribute to a novel and effective treatment strategies for combined gene therapy for lung cancer.
Zhang
Dong-Mei
DM
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Yang
Han-Shuo
HS
Zhu
Wen
W
Feng
Zhi-Hua
ZH
Deng
Hong-Xin
HX
Wei
Yu-Quan
YQ
D000230:Adenocarcinoma / Q000473:pathology; D017209:Apoptosis; D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics*; D008175:Lung Neoplasms / Q000473:pathology*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; C499993:TUSC2 protein, human; D025521:Tumor Suppressor Proteins; D018664:Interleukin-12