| Gastric carcinoma is one of the most common malignant tumors in China, Most of patients with advanced cancer have a poor prognoses due to high recurrence and metastasis,which call for coming out of new therapies .Galoc(1,3)Gal is a carbohydrate epitode,which is a major xenoantigen recongnized by naive antibody in human body, a 1,3-galactosyltransferase transfers galactose from the uridine diphosphate galactose to the N-acetyllactosamine residue on chains of glycolipids and glycoproteins,to form Gala(l,3)Gal,whose terminal a-galactose is specifically recongnized by anti-Gal. Gala(l,3)Gal is produced on the cells of pigs, mice and New World monkey , which is completely absent in humans,apes and Old World monkeys because of inactivation of a 1,3-galactosyltransferase. Lipopolysaccharides and capsular polysaccharide from Gram-negative bacteria contain Gal a (l,3)Gal,and humans body can produce anti-gal epitopeantibodies in response to antigenic stimulation by these bacteria especially colibacillus in gastrointestinal tract. The objective of our study is to clone the gene of murine a 1,3-galactosyltransferase and construct its recombinant eukaryotic plasmid pCDNA3.1/V5-HisA- a 1,3GT. pCDNA3.1/V5-HisA a 1, 3GT was then transfected into GC9811cell by Lipofectamine?000, and the cancer cells expressing a -gal epitope can be lysed by normal human serum through the hyperacute rejection reaction between a -gal and natural antibody. The main methods and results are as following:1. Murine a 1,3-galactosyltransferase was cloned with RT-PCR strategy by using RNA extracted from peritoneal macrophages which were induced with thioglycollate. The PCR product was1221bp in length and testified by DNA sequencing.2. The eukaryotic expression vector pcDNA3.1/V5-HisAa 1,3-GT was constructed by directional cloning the target gene into eukaryotic expression vector pcDNA3.1/.V5-HisA. The direction was confirmed by endonuclease digestion.3. Using liposome mediated method, the human gastric cancer cell line GC9811 was transfected by pcDNA3.1/V5-HisA a 1,3-GT and control plasmid. Stable clones GC9811-a 1,3GT was obtained after G418 screening for two months. The expression of mRNA in tumor cells transfected with GC9811- a 1,3GT was confirmed by RT-PCR.4. Cell proliferation of GC9811-a 1,3GT was inhibited compared with GC9811 cells by using MTT assay.5. The transfected cell expressing Gal a (l,3)Gal was confirmed by indirect fluorescent immuohistochemistry.6. The tumor cell GC9811-a 1,3GT infected with pcDNA3.1/V5-HisA a 1,3-GT highly expressed Gal a (l,3)Gal which was detected and confirmed by flow cytometric analysis.About 97.7 percent of tumor cells GC9811a 1,3GT transfected by pcDNA3.1/V5-HisA a 1,3-GT showed fluorescence in cell membrane,apparently higher than empty vector group (in which only 0.4 percent of tumor cells carry fluorescent) and parental cell group.7. It is showed by human serum killing analysis that 10%-30% cancer cells expressing Gala(1,3)Gal were lysed ,which has a statistics difference compared with untransfected control cells.With increasing of human serum titers ,more cancer cells expressing Gal a (1,3)Gal can be killed.On the other hand, human serum has no significant killing effect on cancer cells of empty vector group and parental group .All these results indicated that gastric cancer cell GC9811 expressing Gal a(1,3)Gal ,which is transfected by a 1,3-galactosyltransferase ,can be killed by human serum. It provides a new idea and method for tumor gene therapy by making a 1,3-galactosyltransferase transfected into gastric cancer cells, which can express Gal(1,3)Gal and be lysed through hyperacute rejection reaction between Gal a (1,3)Gal and natural antibody.
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