| Objective: LIGHT (homologous to lymphotoxins, inducible, competes with HSV glycoprotein D for HVEM, expressed by T lymphocyte), which is the 14th member of tumor necrosis factor superfamily (TNFSF), was found by Mauri and Zhai subsequently in 1998. They reported its molecular mass, locating in chromosome and some biological activities, including stimulating NF-κB dependent translocation, stimulating T cell proliferation and inhibiting the growth of human colon cancer lines HT-29. LIGHT mRNA is highly expressed in spleen, activated T cell and macrophages. PMA and PHA or PMA and ionomycin could induce the expression of LIGHT mRNA in Peripheral blood mononuclear cell (PBMC) in vitro. Similar to other member of TNFSF, the LIGHT protein is a type Ⅱ membrane protein. Cell surface LIGHT can be cleaved by matrix metanoproteinase, forming a soluble protein of 24KD (sLIGHT), which could bind to different receptors expressed on different cells surface and carry on different biological effects. For example, LIGHT could bind to lymphotoxin ( receptor (LT(R) that expressed in many tumor cells surface (such as human breast MDA-MB-231cells, HT-29cells, et al) and induce the apoptosis of tumor cells through caspase-independent pathway; LIGHT could bind to herpesvirus entry mediator (HVEM) expressed in T cell surface leading to activation, proliferation, differentiation of T cell via CD28-independent pathway. In addition, LIGHT palys important roles in the maturation of dendritic cells (DCs), the negative selection of immature T cells, immunomodulation and immunodisease. According to the facts that: (1) LIGHT bind to LT(R that only expressed in tumor cell surface but not or weakly expressed in normal cell and specifically induce the apoptosis of tumor cells; (2) LIGHT bind to HVEM expressed in T cell surface leading to activation, proliferation of T cell which would play important role in the immune response to tumor antigen; (3) sLIGHT gene have the same biological activities as the full LIGHT gene and the sLIGHT is shorter. So it may be more easy to clon and purify than the full LIGHT. We cloned human LIGHT extracellular domain (hsLIGHT) gene, constructed a recombinant expression plasmid pGEX-4T-2/hsLIGHT, and primarily analyzed the condition for the expression of fusion protein GST-hsLIGHT using a prokaryotic expression system, which remains an attractive option for its low cost, more yields and easy purification. The object of this research is to lay a foundation for further studing on its antitumor effects and providing a laboratory experiment base for clinical tumor therapy, exploring new ways of immunotherapy of tumor in the future, and making it possible to develop and produce this biological agent. Methods: Total RNA was extracted from HL60 cells which have cultured in RPMI1640 in the presence of PMA (68 ng/ml) and ionomycin (0.3 μg/ml) for 24 h. The hsLIGHT gene fragment was obtained by RT-PCR with especial primer to hsLIGHT gene. The RT-PCR product was inserted into pGEM-T Easy vector. E.coli DH5α were transformed in Amp 2YT culture medium and the recombinant clones were selected. The positive recombinant clones was extracted and identified by cutting with enzyme and sequensing. The hsLIGHT gene fragment and prokaryotic expression vector pGEX-4T-2 were cut with the same enzymes and a recombinant prokaryotic expression plasmid pGEX-4T-2/hsLIGHT was constructed. E.coli BL21 were transformed in Amp 2YT culture medium and positive recombinant plasmid were selected and incubated in liquid Amp 2YT culture medium until the value of OD600 was 0.5~1.0. Then the expression of the fusion protein GST-hsLIGHT was induced in the presence of IPTG with different concentrations and time; the expression of fusion protein was analyzed by SDS-PAGE; the objective protein hsLIGHT was observed by comparing with different controls and identified by Western Blot. The inclusion bodies and the supernatant of E.coli BL21 lysates which was extracted from 1L culture medium of E.coli BL21 was detected by SDS-PAGE. Expressi...
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