Expression and Detection of Retinol-Binding Protein-4 Gene of Pig in E. coli

다국어 초록 (Multilingual Abstract)

Retinol-Binding Protein-4 (RBP-4) is a low molecular weight lipocalin, which mainly functions as a carrier for vitamin A. Though liver is the main machinery for synthesis of this protein, it is also detectable in other extrahepatic tissues, for example, ovary, uterus, and placenta. Recent evidences have shown that RBP-4 plays important roles in animal reproduction, for example, promoting the development of uterus and embryo. To the best of our knowledge, our laboratory firstly reported that high level of RBP-4 existed in follicular fluid from follicular cysts in sows. Moreover, we have also found that RBP-4 could be secreted by granulosa cells, and RBP-4 receptor was detected in granulosa cells. However, there is no any evidence on the role of RBP-4 in regulating the follicular development. Therefore, cloning and expression of RBP-4 and preparation of polyclonal antibody could help us to explore the role of RBP-4 in follicular development. The aim of this work was to construct prokaryotic expression system of swine RBP-4 gene. The total RNA was extracted from swine’s normal ovarian tissue. The sequence including the whole length of RBP-4 was amplified by RT-PCR and inserted into pEASY-E1.Then transformed into E. coli BL21(DE3)pLysS after gene sequencing. Three hours later, adding IPTG with the final concentration of 1mmol/L and inducing five hours. After centrifugation, the supernatant was discarded. By adding Glucose to Luria-Bertani broth, the expressions of protein were increased. SDS-PAGE showed that the RBP-4 gene expressed in the form of inclusion body with a molecular weight of 21KD. Western-Blot results showed that the target protein could be specifically recognized by mouse anti-human monoclonal antibody. Prokaryotic expression vector of RBP-4 gene was successfully established, and the gene was successfully expressed n E. coli, which is ready for purification and RBP-4 polyclone antibody. Meanwhile, these results were beneficial to investigate the function of RBP-4 in follicular development.