[Cloning, expression and purification of allergen arginine kinase from Periplaneta americana and its allergic activity].
OBJECTIVE
To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity.


METHODS
The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindIII. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA).


RESULTS
The cloned cDNA ORF sequence (Accession no. EU429466) contained 1068 bp and encoded 365 amino acids. Its sequence homology with the published one (Accession no. AY563004) was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 (DE3) as a soluble protein mainly with the molecular weight of about Mr 45000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA.


CONCLUSIONS
The recombinant cockroach arginine kinase has been obtained with proper allergenicity.
Chen
Jia-Jie
JJ
Allergy and Immunology Institute, Shenzhen University, Shenzhen 518060, China.
Xia
Li-Xin
LX
Liu
Zhi-Gang
ZG
Liu
Wen
W
Ji
Kun-Mei
KM
D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D001122:Arginine Kinase / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D015870:Gene Expression; D017344:Genes, Insect; D010526:Periplaneta / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000485:Allergens; D011994:Recombinant Proteins; D001122:Arginine Kinase