[Construction of the recombinant adenovirus vector of interleukin-1 receptor II and its expression in eutopic stromal cells of endometriosis].
OBJECTIVE
To develop a gene therapy vector of interleukin-1 receptor II(IL-1RII) with recombinant adenovirus and express IL-1RII in the eutopic stromal cells of endometriosis(EM).


METHODS
Get full length of cDNA with IL-1RII gene was obtained by PCR. The gene was then subcloned into the pShuttle-CMV shuttle vector. The resultant plasmid (pShuttle-CMV-RII) was cotransduced into E.coli BJ5183 cells with pAdEasy-1 plasmid to undergo homologous recombination by electroporation. The linearized recombinant plasmid (pAd-RII) was transfected into 293 cells. The recombinant adenovirus was detected by examining the expression of IL-1RII while the recombinant adenovirus of LacZ gene was constructed as control. The stromal cells of EM were infected by the recombinant adenovirus and the expression of IL-1RII was detected by immunohistochemistry(IHC).


RESULTS
It was confirmed by sequencing that the two ligand products had no mutation of IL-1RII. Restriction endonuclease analysis confirmed the successful cloning of the gene into the pShuttle-CMV and the recombinants(pAd-RII) were selected for kanamycin resistance. Presence of the recombinant adenovirus was confirmed by the X-gal stain of LacZ and the expression of soluble IL-1RII was detected by ELISA. IL-1RII was expressed in the stromal cells of EM by IHC.


CONCLUSIONS
The recombinant adenovirus of IL-1RII has been successfully constructed and expressed in the stromal cells of EM, which provides a basis for the research into the role of IL-1RII and the effect of biological treatment in EM.
Center of Clinical Reproductive Medicine, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. zhhou1981@126.com
Zhen
Z
Hou
Jing
J
Zhou
Li-ying
LY
Gao
Yan-qiu
YQ
Hu
Jia-yin
JY
Liu
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D004715:Endometriosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology* / Q000628:therapy; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D016133:Polymerase Chain Reaction; D053574:Receptors, Interleukin-1 Type II / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D004274:DNA, Recombinant; D053574:Receptors, Interleukin-1 Type II; D004262:DNA Restriction Enzymes