Cloning, Sequence Analysis And Expression Of SOCS-2 CDNA In Pig

Adipose tissue is composed of a sum of adipocytes. Growth of adipose tissue is the result of proliferation and differentiation of preadipoctes as well as the enlargement of mature adipocytes.Adipogenes is a result of the regulation of lipases at the transcriptive level. The distinct characteristic of cytokines working fashion is strictly regulated in temporal and spatial. There are at least three kinds of protein families involved in the negatively regulating the cytokine signaling: SOCS, PIAS and SHP-1. Among them, the study of the SOCS family is relatively extensive. SOCS-2 is one of the main members of the SOCS family. Many experiments have proved that SOCS-2 can regulate several cytokine signaling in vivo or in vitro.Among them, the GHR signaling is the most important, which regulate the growth and development of organism.Therefore, SOCS-2 would be an important regulator during the growth of muscle tissue and metabolism of lipids.In this study ,the porcine SOCS-2 cDNA has been cloned . Then, we analysised the DNA sequence, the structure of predictioned protein and expression of SOCS-2. The main results were summarized as follows:1. The cDNA sequence of SOCS-2 gene was cloned by RT-PCR firstly (GenBank accepted number is EF121242). Thereafter, It was inserted into PMD19-T vector by T/A cloning , transformed into DH-5α, tested by PCR and sequenced . The data showed that the homology of the cloned porcine SOCS-2 , including 822 bp ,was more than 93% and that of the deduced amino acid sequence was 89% when compared with human, rat and mice . And the molecular weight of SOCS-2 protein is about 22.25KD and PI is 8.03.By the CDD analysis, it has the complete SH2 and SOCS-box.2. The tissue distribution analysis showed that SOCS-2 mRNA could be detected in many tissues extensively, such as heart, lung, subcutaneous adipose, abdominal adipose, liver,spleen, kidney and muscle. Its expression was higher in kidney and heart but lower in muscle tissue.It is coincidence with its portant role in the regulation of growth and development organism by GHR signaling.3. The SOCS-2 has been cloned by RT-PCR. Digested by BamHⅠa nd HindⅢ, the SOCS-2 and pET-43a vector were connected by the T4-DNA ligase.Then , recombinated pET-43a-SOCS-2 was transfected into Ecoli.BL21.Identifited by PCR,digested by BamHⅠa nd HindⅢand sequence ,the positive pET-43a-SOCS-2 was induced by IPTG.The data showed that the we successfully expressed in the BL21.4. SOCS-2 mRNA can be detected during whole differentiation process of porcine preadipocytes in vitro.The SOCS-2 mRNA rised time-dependly from the second day when the culture cell start to confluence.And the expression of marker genes of adipocyte, such as LPL,HSL,PPAR-γ2 ,upregulated in the same way.However ,the data showed it wasn't significant by t-test .It could be explained that SOCS-2 may not regulate the differentiation of preadipocyte .