Expression, Purication And Functional Analysis Of The Gene PGRMC2 From Silk Worm, Bombyx Mori

According to the large-scale cDNA sequencing of silkworm pupae, a unique new Bombyx mori cDNA was identified after Blasting toward NCBI database. The novel gene contains a PGRMC2 domain, so we preliminary nominate it as PGRMC2 (Bombyx mori PGRMC2), and the GenBank accession number is DQ311390. The length of the gene is 772 bp. It contains an ORF of 681 bp and encodes 226 amino acids with the predicted molecular weight of 29.054 kD and isoelectric point of 5.67.According to the ORF of PGRMC2, two primers were designed to obtain the coding region of PGRMC2 gene from silkworm pupae EST database. The ORF of the gene was cloned into pET-28a vector digested with EcoR I and Xho I. The recombinant plasmid was transformed into E.coli Rosseta (DE3). PCR and digestion with EcoR I and Xho I showed that the designed fragment was inserted correctly. The recombinant plasmid was sequenced and the sequence map indicated that a recombinant expression plasmid was constructed successfully. Recombinant protein was expressed successfully in E.coli Rosseta (DE3) induced by IPTG with the final concentration of 1mmol/L. The analysis of SDS-PAGE showed that the fusion protein His-PGRMC2 was expressed highly in Rosseta with 29 kD that was accorded with the theory value. The most recombinant protein was soluble and was purified with metal-chelating affinity chromatography. Polyclonal antibodies were generated by immunization of New Zealand rabbit with recombinant protein. Polyclonal antibodies were prepared by affinity chromatography using immobilized protein A and the titer was larger than 1:25600, measuring by ELISA. Western blotting analysis showed that the antibody could bind His-PGRMC2 specifically. Some tissues of the fifth instar larvae, such as brain, epidermis, silk gland, gut, Malpighian tubule, fat body, stigma were extracted and analyzed by Western blotting. We also compared the quantity of PGRMC2 mRNA in different tissues of the fifth instar larva and different stages of silkworm. These results suggested that PGRMC2 highly expressed in silkworm's moth, seminal glands. Immunocytochemistry in BmN cells showed that the protein mainly existed in the cytoplasm and membrane. These results laid a good foundation of further studies of PGRMC2.