Cloning And Expression Analysis Of Vitellogenin Receptor Gene From Actias Selene Hubner

Actias selene Hubner(Lepidoptera Saturniidae) is a kind of undeveloped wild silk producing insect and mainly lived in China, Japan, India and East and Southeast asia country, Belonging to Lepidoptera Saturniidae, scientific name, Actias Selene Hubner. Distributed in East and Southeast asia country. Few reports about it were found and most of them are on the morphology and biological behavior.During insect oogenesis, developing oocytes of oviparous animals accumulate large amounts of vitellogenin (Vg), the extra-ovarian yolk protein precursor, vitellogenin synthesised by the fat body and secreted into the body fluids. The selective internalization of Vg by the growing oocyte is achieved through receptor-mediated endocytosis. Therefore, the researches on biochemical characteristics and molecular characteristics of receptor become research popular.VgRs are belong to the low-density lipoprotein receptor (LDLR) superfamily (Willnow et al.,1999) and have common structural elements like:(i) the ligand-binding domain comprising Class A cysteine-rich repeats;(ii) the epidermal growth factor (EGF) precursor homology domain containing Class B cysteine-rich repeats and YWXD repeats;(iii)an O-linked carbohydrate domain;(iv) a transmembrane domain;and (v) a cytoplasmic tailPrimers were designed by Primer premier5.0software according to VgR sequences from Bombyx mori and other insects, oligonucleotide. A cDNA fragment of6270bp was obtained by RT-PCR and RACE-PCR. The sequence had been deposited in the GenBank database with accession number JQ809472. Nucleotide sequence analysis revealed that VgR cDNA contains a560bp5’-untranslated sequence, a putative ORF of5439bp, a271bp3’-untranslated region (3’UTR) and a putative polyadenylation signal. Sequence encoding a1813amino acid protein with a molecular weight of203.9kDa. Based on the deduced amino acid sequences, the structure of the Ash-VgR is indicated by using the ExPASy Proteomics tools. Phylogenetic analysis indicated that Bombyx mori gene has98.2%identity with Actias selene Hubner VgR.The functional domains of vitellogenin receptor from Actias selene were analyzed using the ScanProsite software and two ligand binding domain of LBD1and LBD2were selected for prokaryotic expression.The ORFs of two domains of Ash-VgR were amplified by PCR and ligated to pET-28a vector for protein expression. Two recombinant proteins with a molecular weight of about24kDa and40kDa were detected by SDS-PAGE and the expression were not influenced by different IPTG concentrations. Western blot analysis of recombinant protein showed that a consensus24kDa and40kDa protein band was detected using anti-His antibody, while there was none in the control group. All this indicate the successful expression of the recombinant VgR proteins in E.coli BL21(DE3) cells. This will provide the foundation for further study on eukaryotic expression and function of vitellogenin gene.