Construction Of The Expression Plasmid Of Human Polypeptide: N-Acetylgalactosaminyltransferase 2 Gene Sense And Antisense RNA

Human ppGalNAc-T2, a member of polypeptide N-Acetylgalactosaminyltransferase ,is a type Ⅱ membrane protein of about 64.7kD encoding 571 aa. Human polypeptide N-Acetylgalactosaminyltransferase 2 ,purified from human placenta by White and Clauson in 1995,cloned from λgt10 cDNA of human stomach cancer cell on the basis of its part aa. It located at 1q41 -q42 with the length of 1713bp,which is widely distributed in various tissues, especially in the mucin secreting tissues and some organs as colon, small intestine, stomach, pancreas, skeletal muscles, testicle and ovary. Polypeptide: N-acetylgalactosaminyltransferase is a glycosyltransferase involved in the first step of O-glycan synthesis. It catalyzes the transference of GalNAc group from UDP-GalNAc to the threonine (Thr)or serine (Ser) residue within a definite sequence on protein polypeptide chain to form a GalNAc α-O Thr/Ser glycosidic linkage.Tumor cells have some mucin-like glycoproteins, which may play an important role in invasion, metastasis and growth of tumor cells. Mucin-like proteins have many O-linked glycosylation domains, so ppGalNAc-Ts as the first enzyme of mucin-like O-linked glycosylation must play an important role in the occurrence and development of tumour. ppGalNAc-T2 has a widely distribution and substrates spectrum in its family, so it is perhaps the rate-limiting enzyme of O-glycosylation ,having an important biological significance So ,it is chosen to be studied in this article.1. Study on expression difference of ppGalNAc-T2 in tumor cells on mRNA level and expressive spectrum of mRNA of ppGalNAcT family in two tumorcellsWith RT-PCR we examined the expression on mRNA level of ppGalNAc-T2 in different tumour cells. The result indicated that ppGalNAc-T2 is widely expressed in tumor cells, it is at different mRNA levels in glioma cell Line SHG44, stomach cancer cell Line SGC7901, leukemia cell Line SHI-1, Lung Cancer Cell Line A549 and ovarian cancer cell line HO8910. With P-actin as the internal standard, the result showed that the expression of ppGalNAc-T2 is low in glioma cell Line SHG44, while in stomach cancer cell Line SGC7901 it is very high, but in fibroblasts cell line NIH3T3 there is no expression or lower expression. With the same method we detected the mRNA expression of ppGalNAcTs in two kinds of tumor cells (SHG44 and SGC7901).The result showed that ppGalNAcT2,T6,T8,T10,Tll,T13 were expressed in different level in SHG44 cell and ppGalNAcT2,T4,T7,T8,Tll,T13 were expressed in different levels in SGC7901 cell.2 Construction of Human ppGalNAc-T2 Gene Expression Plasmid and ItsEffect on Tumor Cell SHG44With PCR the target DNA fragment enconding ppGalNAcT2 was amplified from cloning plasmid pDONR201-T2 and subcloned to the eukaryotic expression plasmid pcDNA3.1. The recombinant plasmid was transfected into SHG44 cells, then was screened by G418. The high expression of ppGalNAc-T2 cell was detected by RT-PCR and western blot. We also assayed the mRNA of MMPs^ TGF-01 with RT-PCR, these genes play an important role in invasiveness, metastasis and growth of human tumour cells. With MTT and flow cytometric we examined the varieties of cell proliferation and cell cycle. The results showed that the mRNA of MMPs ,TGF-pl decreased ,but the mRNA of MMP14 and UPAR increased while expression of ppGalNAc-T2 in cell was higher .The results also showed that the higher expression of ppGalNAc-T2 cell grew faster.3 Construction of Human ppGalNAc-T2 Gene Antisense RNA ExpressionPlasmid and Its FunctionsWith PCR two different length ppGalNAcT2 gene segments were amplified from cloningplasmid pDONR201-T2 and subcloned to the eukaryotic expression plasmid pEGFP-Cl,Then the recombinant plasmid was transfected into SGC7901 cells. A series of subcellular clones were aiming at the blocking of ppGalNAcT2 gene expression of SGC7901 cells were established by G418 . After antisense blocking of ppGalNAcT2 gene expression were tested by use of flow cytometr and fluorescent microscope , the variational characteristics of MMP2 and TGF-pl mRNA and cells growth were studied with RT-PCR.The cell proliferation was examined by MTT. And the shape changes of the cells were also observed by microscope . Laser scanning confocal microscope utilized monitor the distribution of green fluorescence(GFP).The results of RT-PCR revealed that the blocking of ppGalNAcT2 gene expression could increase the mRNA level of MMP2 and TGF-pi but decrease that of MMP14 and UPAR. MTT result indicated that the blocking of ppGalNAcT2 gene expression had influence on GC7901 cell proliferation.The result also showed that the transfected cells' shapes had been changed and the green fluorescence distributed in cytoplasm.