cDNA cloning and recombinant expression of the general odorant binding protein II from Spodoptera litura.
A cDNA encoding the general odorant binding protein II (GOBP II) was isolated from the antennae of Spodoptera litura (SlGOBP II, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SlGOBP II was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72. SlGOPB II shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SlGOPB II shared significant identity with the GOBP II from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SlGOBP II was specifically expressed in the antennae. cDNA encoding SlGOBP II was constructed into the pET-32a vector and the recombinant protein was highly expressed in Escherichia coli BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPII i.e, 32 kD, which has a 6xHis tag at the N-terminus. The recombinant SlGOBP II was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1:12800, while Western blot analysis showed that the recombinant SlGOBP II was recognized as anti-SlGOBP II antiserum.
Jin
FengLiang
F
Engineering Research Center of Biological Control, College of Natural Resources and Environment, South China Agricultural University, Ministry of Education, Guangzhou, 510642, China.
Dong
XiaoLin
X
Xu
Xiaoxia
X
Ren
ShunXiang
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010802:Phylogeny; D011817:Rabbits; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D018411:Spodoptera / Q000737:chemistry / Q000235:genetics*
D000906:Antibodies; D018076:DNA, Complementary; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein