Silkworm 30kd Set Out The Preliminary Study Of The Expression And Function Of Lipoprotein 19g1

The 30K protein group have common characteristics in amino acid composition and immunological activity. They are a group of structurally related proteins with a molecular mass of approximately 30 kD. 30K proteins are not female specific lipoproteins, which are synthesized in the fat body of silkworm. Furthermore, it was found that expression of these lipoproteins is regulated by juvenile hormone. They are the main storage proteins used for growth of silkworm and the major protein components of the hemolymph of the fifth instar larvae and of pupae. In female, 30K proteins are the major component of the yolk protein. Among the 30K protein, there are more researches on 30 kD lipoprotein 6G1 and 19G1. The 30Kc6 gene was cloned into expression vector to produce this protein in Escherichia coli, and the protein was purified. The purified 30K protein effectively inhibited insect cell and mammalian cell apoptosis by addition to culture medium. An interferon-b producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed. The results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.The EST of low molecular mass 30 kD lipoprotein 19G1 precursor (GenBank accession no. AY568957) was found through searching our cDNA bank of silkworm pupae. By means of bioinformatic methods, the ORF contains no intron but an exon of 771 bp encoding 256 amino acids; the predicted molecular weight is 29.5 kD and the pI is 7.290. The ORF of the gene was cloned into the prokaryotic expression vector pET-28a(+) and the recombinant plasmid was transformed into E.coli BL21(DE3). The fusion protein was successfully expressed,purified and then immuned New Zealand rabbit to get antiserum. The titer of the polyclonal antibodies reaches 1:12800 measured by ELISA. We compared the quantity of 30Kc19 mRNA in different tissues of the fifth instar larva and different stages of silkworm using RT-PCR. And we demonstrated that apoptosis is inhibited by supplementing the culture medium with this purified recombinant 30K protein.