[Cloning of the intact encoding gene of human DC-SIGN and prokaryotic expression of its extracellular region].
OBJECTIVE
To obtain the intact encoding gene of human DC-SIGN and express its extracellular region in E.coli.


METHODS
The intact cDNA encoding human DC-SIGN was amplified from total RNA of placenta of healthy parturient by RT-PCR, and its extracellular region was inserted into prokaryotic expression vector pET-41a. The recombinant plasmid pET-41a-sDC-SIGN was transformed into E.coli BL21(DE3). The expressed product was purified by GST affinity chromatography and identified by SDS-PAGE and Western blot.


RESULTS
The DNA fragment of about 1 300 bp was amplified by RT-PCR, and cloned into pGM-T vector to obtain the recombinant plasmid pGM-DC-SIGN. The DNA fragment encoding the extracellular region of human DC-SIGN was amplified from pGM-DC-SIGN plasmid and the recombinant expression vector pET-41a-sDC-SIGN was constructed. The component of M(r); 66 000 in the purified recombinant product was found to be recognized by anti-DC-SIGN antibody.


CONCLUSIONS
The cDNA of human DC-SIGN is cloned and the protein of its extracellular region is obtained successfully, which lay the foundation for further research on functions of DC-SIGN.
Liu
Ying
Y
Department of Immunology, Southern Medical University, Guangzhou, China.
Zuo
Da-ming
DM
Lu
Xiao
X
Zhang
Li-yun
LY
Chen
Zheng-liang
ZL
D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D000906:Antibodies; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D037181:Lectins, C-Type; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin