The Clone Of Human Mature TGF-β₁ And Its Expression And Clinical Detection Of Serum TGF-β₁ By Using Its Polyclonal Antibody

Objective: It is known that TGF-β1 is the most important cytokine which can cause hepatic fibrosis. The relationship between the serum TGF-β1 and hepatic fibrosis as well as its role for early diagnosis of the liver fibrosis have been payed more and more attention by the people. The aim of this study was to clone a human mature TGF- β1 gene and to obtain fusion protein of TGF- β1 through the expression of this gene, then to get the rabbit-anti-human TGF- β1 polyclonal antibody from the immune rabbit with this fusion protein. Furthermore, we detected the serum TGF-β1 with this polyclonal antibody in clinical patients.Methods: At first, we separated the peripheral blood mononuclear cell (PBMC) from the patient with chronic hepatitis B and cultured it for 48 hours. Then we extracted the total RNA from the PBMC. Using the RT-PCR, we got the fragment of human mature TGF-β1 gene with the coding region, then the target gene was cloned into an eukaryotic expression vector pMAL-C2X by using the recombinant DNA technique and it was confirmed by DNA sequence analysis and by restriction enzyme digestion. After induced by IPTG, the fusion protein MBP-TGF-β1 was expressed in JM109 E.coli. The fusion protein was purified by affinity chromatography using amylose resin and testified its antigencity by Western-blot. Secondly, we immunized two rabbits with Img and O.Smg of the purified fusion protein emulsified with an equal volume of Freund's immunoadjuvant. After the rabbits were injected with the purified fusion protein as immunogen for 6 times, we collected theimmune sera of rabbits. After we separated the sera from the blood and the antibody (IgG) from the immune sera were purified by ammonium sulfate fractionation,then the parified antibodies of the rabbit-anti-human TGF-β1 polyclonal antibodlies were obtained with the purified antibodies, we used the indirect sandwich enzyme immunoussay to detecte the serum TGF-β1 in 32 samples of patients with chronic hepatitis B or cirrhosis and 8 normal sera as controls. The results were compared with the human TGF- β1 EIA kit of foreign standardized products.Result: The results of our experiments were as follows:1. We obtained the gene fragment of human mature TGF- β1 which containes the coding region, and was identified by DNA sequence analysis.2. We constructed the clone of the recombinant pMAL-C2X- TGF-β 1/ JM109 and its expression of fusion protein MBP- TGF- β1 with antigenicity was confirmed by using the Western-blot analysis.3. We obtained the purified fusion protein MBP- TGF-β1 by using the affinity chromatography method and it was identified by SDS-PAGE analysis. Then we used the purified fusion protein as immunogen to immunize the rabbits.4.The purified specific rabbit-anti-human TGF- β1 polyclonal antibodies were obtained from the immune sera of rabbits, and it can react with human TGF- β1.5. The level of serum TGF- β1 was increased in patients with chronicIVhepatitis or liver cirrhosis as compared with normal controls (P < 0.05). It suggested that the level of serum TGF- β 1 was gradually rising in the course of chronic liver disease. The levels of serum TGF-β1 from patiants that were detected with our TGF- β 1 polyclonal antibodies were similar to the levels of serum TGF- β1 that were detected with the standard EIA kit.Conclusion: The fusion protein MBP- TGF- β1 that was expressed by the recombinant pMAL-C2X-TGF- β 1/JM109 has immunogenicity. The TGF-β1 polyclonal antibody was obtained from the immune sera of rabbits. The rabbit-anti-human TGF-β 1 poly clonal antibody can be developed an ELISA kit for detecting the serum TGF-β1 and may be used for the diagnosis of initial stage of hepatic fibrosis.