Molecular cloning of cDNA coding for the 68 kDa allergen of Penicillium notatum using MoAbs.
To characterize the 68 kDa allergen of Penicillium notatum (also known as P. chrysogenum), a molecular antibody (MoAb) (P40) was previously generated. For cDNA cloning, three more MoAbs (3F, 5A3, 5G2) were generated in the present study. A mixture of all the four MoAbs was used in cloning of the gene coding for the 68 kDa allergen from a lambda gt11 cDNA library of P. chrysogenum. A cDNA clone (A6) with DNA insert of about 0.5 kb which encodes for the 3'-terminal nucleotide sequence of the 68 kDa allergen was obtained. The cloned sequence contained two putative N-glycosylation sites. The reduction in molecular weight from 68 to 62 kDa in immunoblotting after treatment of the crude extract of P. notatum with N-glycosidase F indicates that the 68 kDa allergen is a glycoprotein. Nucleotide sequence determination showed that 188 (54%) of the 348 nucleotides of the cDNA sequence obtained were identical to the same region of the nucleotide sequence of the beta-N-acetylglucosaminidase gene of Candida albicans. Although the cDNA clone obtained did not encode the full-length gene of the 68 kDa allergen, polypeptide expressed from the A6 cDNA showed positive immunological reactivities to all four MoAbs used in the cloning experiment and to IgE antibodies in sera of asthmatic patients. There was a loss of immunoblotting activity to the 68 kDa component after absorption of MoAb P40-containing culture supernatant with filters blotted on plaque lawns of cDNA clone A6. Moreover, the immunoblotting activity remained when the MoAbs affinity-purified with filters containing polypeptides encoded by the cDNA insert of clone A6 were used.(ABSTRACT TRUNCATED AT 250 WORDS)
Shen
H D
HD
Department of Medical Research, Veterans General Hospital-Taipei, Taiwan, Republic of China.
Liaw
S F
SF
Lin
W L
WL
Ro
L H
LH
Yang
H L
HL
Han
S H
SH
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010407:Penicillium / Q000276:immunology*
D000485:Allergens; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary