Expression And Purification Of Galectin-1 And Anti-tumor Research

Galectin-1, also called as LGALS1, is a member ofβ-galactoside-binding proteins. To date, 14 mammalian galectins have been identified. Different from other lectins, each member of galectin family contains at least one domain of about 130 amino acids, which is responsible for the observed carbohydrate-binding activity, and therefore is named as the carbohydrate recognition domain(CRD). Recent observations suggest that galectin-1 can crosslink some glycoconjugates receptor and trigger a cascade of transmembrane signalling events, contributing to cell adhesion, cell development, cell apoptosis and inflammation response under physiological or pathological condtions.Initially, galectin-1 could play an important role in immune system. Galectin-1 induces apoptosis of human T cells and anti-inflammatory response. Through binding to membrane protein, galectin-1 can also suppress microbial infection. Subsequently, galectin-1 may involve in transformation and development of tumors. It participates in the initiation of transformed phenotype of tumors. By interacting with integrin molecules or regulating its expression of integrin molecules, galectin-1 can control the invading progression into tumor. It can induce apoptosis of lymphocyte invaded by tumor. Galectin-1 secreted by tumor cells can also regulate of tumor angiogenesis.In order to study the function of galectin-1, galectin-1 gene was cloned from recombinant vector pcDNA3.1-Gal-1 by PCR. The amplified fraction was cloned into pGEMT to construct recombinant plasmid pGEMT-Gal-1.When the recombinant vector pGEMT-Gal-1 was obtained and identified by sequence validation, the correct sequence was ligated into prokaryotic expression vector pET21a(+) and thus recombinant plasmid pET21a(+)-Gal-1 was acquired successfully. Then, the recombinant vector pET21a(+)-Gal-1 was transfered into BL21(DE3). Induced by IPTG, the recombinant protein was efficiently expressed in the E.coli BL21(DE3). SDS-PAGE and Western blot indicated that the expressed protein was Gal-1. Because the recombinant protein was combined with six His tag, Ni column was used to purify expression products. The purity of recombinant protein can reach more than 85%. The concentration of purified protein was detected and the activity of galectin-1 was determined by hemagglutination assay using mouse crythrocytes. It indicated that the activity of recombinant protein was fine.Although the protein combined with His tag at C-terminal possessed high purity and good activity, it would deposit at next day. Account of methods was used to improve the solubility of the recombinant protein, such as dialysised immediately, changed the solution buffer, addedβ-ME or glycerol, and added PEG to the recombinant protein. But no evidence effect was achieved. The recombinant protein was soluble just at a low concentration. Considering GST can enhance the soluble of galectin-1, like the method used above, galectin-1 gene was cloned again and ligated into vector pET42a(+) contain GST and His tag. Then, the recombinant plasmid pET42a(+)-Gal-1 containing correct gene sequence was achieved, subsequently, the recombinant vector pET21a(+)-Gal-1 was transferred into BL21(DE3). Induced by IPTG, the recombinant protein galectin-1 was efficiently expressed in the E.coli BL21(DE3) analyzed by SDS-PAGE. Ni column was utilized to purified recombinant protein, the purity of recombinant protein can reach more than 85%. GST column was utilized to purified recombinant protein farther more. The results showed that the solubility of galectin-1 increased and protein activity was enlarged 5~6 times.To study on how galectin-1 influence tumor cells grow in vivo and in vitro, RNAi technology was selected to suppress expression of galectin-1 in the cell line which had determinated galectin-1 expression. Then observe the change of cell cycle and how it influences the growth of tumor cell in nude mouse.At first, several human tumor cell lines was analyzed whether its express galectin-1 or not by RT-PCR, then some cell lines was proved to express galectin-1 assistant detected by Western blot. Because nude mouse can easily formed tumor when Hela was injected, so Hela cell line was selected for farther researching. According to the character of siRNA, two sequence of galectin-1 was selected as target sites. Complement oligonuclear was designed and synthesized for each target sites, and ligated into pSilencer after denaturing and annealing to achieve pSilencer-RNA-1 and pSilencer-RNA-2 respective. Vector pSilencer-RNA-1 and pSilencer-RNA-2 was constructed successfully and identified by sequence validation. Then pSilencer-RNA-1 and pSilencer-RNA-2 was transformed into Hela cell by electrical transformation. Anti-hygromycin clone was selected using 400μg/ml Hygromycin B, and detected by RT-PCR subsequently. Several clones cannot be detected showing that the gene was silenced. Control cell was Anti- hygromycin clone when pSilencer was transformed.To investigate how did the tumor was influenced when the galectin-1 was decreased in cell, MTT assay was used to demonstrate that the cell growth status has no influence when tumor cell was cultured in vitro. Hela cell which galectin-1 gene was silenced was injected into nude mouse neck to observe the change of tumor growth for three weeks continuous. The result showed that the weight of group which injected tumor cell with galectin-1 gene silencing is lighter than the control group. Analyzed by statistics software, the differences are remarkable. The results illuminated that tumor growth was suppressed when galectin-1 gene was suppressed. So, galectin-1 can be a potential cancer target for cancer therapy. This experiment can be a good basic for next study.