Expression Of TGF-βⅡ Receptor Extracellular Domain In E.Coli

Objective: Transforming growth factor β1(TGF β1) plays an important role in a broad spectrum of cancer cell functions, such as growth, differentiation, apoptosis, extral-cellular matrix formation, neovascularization and immunosuppression. TGF β1 is known as a famous growth-inhibitor for normal epithelial cells, however, a number of human cancer resist the inhibitory effect, and TGF 3 might facilitates tumor cell invasion and metastasis. Because tumor-associated TGF 3 can cause immune suppression. So inhibiting the function of TGFβis the key-point that enhances anti-tumor immune responses. Recently, there are many studies tried to blocking the immune-suppressed caused by TGFβ. Including that using anti-TGFβ antibody to counteract TGF 3 , or using antisense nucleic acid technique to block TGF 3 . But these measures can not get the satisfactory outcome. If we can inhibit secretion of TGF β and the function of immune-suppression, it will be in favor of controlling the progression of tumor, and improving the outcomes. The expressed soluble type II transforming growth factorβ receptor can absorb the over-expressed TGFβ , thus it can efficiently occlude the bio-activity of TGF- β, break the immune-inhibitory caused by tumors. Materials and methods: According to extra-cellular domain cDNA sequence of T β RII, we synthesized the fragment. Extra-cellular domain gene was amplified by PCR, and cloned to the pGEM~T easy plasmids vector. The inserted fragment was cut by EcoR I and Xho I enzyme, released from pGEM-T-easy-T β RII plasmids vector. The recombinant extra-cellular domain cDNA sequence of TβRII was linked with pGEX~4T-3 plasmids byblunt end ligation., then we gained the T P RII/pGEX-4T~3 vector, and it was transfered to E.coli and amplified. After the evaluation by PCR, and sequencing, the recombint pasmids were induced expression , fused protein GST-T 0 RII purified by GSH桝garose. Analyzed by SDSPAGE western blotting. We detected the lymphocyte activity associated with TGF-P and TPRII, observed the bio-activity in vitro.of the expressed production. Results: We amplified the 499bp fragment. The sequence of inserted fragment was correct in pGEM-T easy vector , evaluated by sequencing. The size and location of recombint vector pGEX-4T~3~T P RII was correct, evaluated by sequencing. SDS-PAGE electrophoresis analysis showed that a new band appeared at the 54KD region, it corresponded to theoretical value. So we could consider that the pGEX-4T-3-T P RII had secreted the aimed protein. Western-blotting analysis also confirmed that recombint pGEX-4T-3-T 3 RII expressed T {3 RII protein. We observed the rescue effect of recombint TJ3RII protein, used the rhTGF 0 1 as inhibitor. The result showed that the recombint TPRII protein could relieve the lymphocyte activity inhibited by TGFP1. Dicussion: We constructed T P R II/pGEX-4T-3 expression vector, and transferred it into BL21(DE3) E.coli. succeeded expressing recombint protein GST-T3 RII, and the protein represented the effect that could relieve the lymphocyte activity inhibited by TGF01.