[Construction of double-subunit co-expression eukaryotic vector of human interleukin 12].
OBJECTIVE
To construct the double-subunit co-expression plasmid P(+)/IL-12 to fulfill the gene therapy.


METHODS
The full length cDNAs of P40 and P35, two subunits of hIL12, were amplified from the reverse transcription production of human embryo kidney using polymerase chain reaction (PCR) and respectively cloned into eukaryotic expression vector pcDNA3.1(+/-) to construct the single-subunit plasmids--P(+)/P40 and P(-)/P35. Then we linked cDNAs of P40 and P35 tandem and cloned them into pcDNA3.1(+) to get P(+)/IL-12 plasmid. The hIL-12 protein in supernate was detected with enzyme-linked immunoabsorbent assay (ELISA) after P(+)/IL-12 was transfected into HepG2 through liposome.


RESULTS
The plasmid of P(+)/IL-12 was testified using enzyme cutting and sequencing analysis. ELISA showed that hIL-12 protein could be expressed after transfecting into HepG2.


CONCLUSIONS
The successful construction of P(+)/IL-12 plasmid can not only simplify the operation steps but also mimic hIL-12 physiological expression style in the gene therapy, thus laying the foundation for the use of hIL-12 in the gene therapy.
Yang
Dong-ye
DY
Department of Gastroenterology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Lu
Fang-gen
FG
Zhao
Shui-ping
SP
D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics*; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
C508858:IL12A protein, human; D053765:Interleukin-12 Subunit p35; D053762:Interleukin-12 Subunit p40; D021122:Protein Subunits; D011994:Recombinant Proteins; D018664:Interleukin-12