[Cloning, expression, purification and identification of Der f6 gene and its immunological characteristics from the dust house mite].
OBJECTIVE
To construct, purify and characterize a recombinant expression plasmid containing Der f6 gene of Dermatophagoides farinae.


METHODS
A pair of primers was designed according to the known sequence of Der f6 gene. The live mites identified and cultured locally were picked and the total RNA was extracted. The Der f6 gene fragment was amplified by RT-PCR, and cloned into pMD18-T vector, and then transferred into E. coli Top10. The target gene obtained from the recombinant plasmid by digestion with EcoR I and Xho I was connected to the prokaryotic expression vector pET-24a. The expressed recombinant plasmid containing Der f6 gene was constructed by cloning target gene into pET-24a and first transferred into E. coli Top10, then into E. coli B121 (DE3). The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting, and was purified by immobilized metal ion affinity chromatography (IMAC).


RESULTS
The two recombinant plasmids, pMD18-T-Der f6 and pET24a-Der f6, were constructed. SDS-PAGE showed a correct molecular weight of the recombinant Der f6 protein. After purification by affinity chromatography, the protein showed only one strip on SDS-PAGE gel and appropriate combination ability with IgE in sera of allergic patients.


CONCLUSIONS
The Der f6 gene has been cloned into plasmid pMD18-T vector and sub-cloned into the expression vector pET-24a, the recombinant plasmid pET24a-Der f6 has been expressed in E. coli BL21 (DE3), purified by IMAC, and showed appropriate IgE-combined ability.
Zhu
Yong-feng
YF
College of Life Sciences, Shenzhen University, China.
Liu
Zhi-gang
ZG
Gao
Bo
B
D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D001249:Asthma / Q000097:blood / Q000469:parasitology*; D001483:Base Sequence; D003001:Cloning, Molecular; D040001:Dermatophagoides farinae / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D010957:Plasmids; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012221:Rhinitis, Allergic, Perennial / Q000097:blood / Q000469:parasitology
D000485:Allergens; D007073:Immunoglobulin E; D012313:RNA