Expression Of The Helicobacter Pylori γ-glutamyl Transpeptidase (ggt) Gene And Effect On Cell Growth

Helicobacter pylori (H. pylori) is a spiral, gram-negative and microaerophilic bacterium, and is one of the major factors inducing pathological changes in human gastric mucosa. One half people in the world are infected with it. The virulence factors of H. pylori include Cytotoxin-associated gene A (cagA), Vacuolating cytotoxin A (VacA) and Urease (Ure), etc. Recently, y-glutamyl transpeptidase (ggt) was found to be related to Pathological process.Analysis of the ggt gene structure indicated that the open reading frame (ORF) was 1,704 bp, encoded 567 amino acids, according to the information of the ggt gene published in GenBank web site (National Center for Biotechnology Information). The result of functional structure prediction showed that there is a 30aa hydrophobic region in the N-terminal of the protein, the 80aa in front of the protein was the leader peptide, and the enzymic activity center was at C-terminal after 380 animo acids. To further insight into the relationship between function and the structure of the ggt gene, we cloned the functional regions of the gene and analyzed, and obtained the results below:(1) construction of the recombinant baculoviruses expressing the ggt fragments:According to the sequence of ggt gene published in GenBank, we designed the specific primers to amplify the fragments, including that of the full length of the ORF (ggt-all), deletion of N-terminal 30aa (ggt-d30a), deletion of N-terminal 80aa (ggt-d80a), and deletion of N-terminal 380aa (ggt-d380a). The sequencing result showed that none of the no-sense mutations occurred. The amplified fragments were inserted into the bculovirus transfer vector pFastBacl after the fragments were digested with restrict enzymes. The vector DNA was transformed into receptor bacteria BmDH10Bac, and the recombinant viral DNAs were obtained, respectively. The DNAs were transfected into the insect BmN cells. The recombinant viruses were obtained, named as BmNPV-ggt, BmNPV-d30a, BmNPV-d80a, and BmNPV-d380a. As same, the viruses expressing fusing gene with gfp were constructed. The inserts were confirmed by amplification of the gene fragments.(2) Product activity and cellcular location of the ggt fragmentsActivity of the ggt fragment products was analyzed after the cells infected with the variant viruses were collected. The results showed that the activities of the expression products of BmNPV-ggt-all, BmNPV-ggt-d30a, BmNPV-ggt-d80a, and BmNPV-ggt-d380a reached 3.61,10.50,10.03和10.88 U/L, respectively, and the control of BmPak6 was of 0.06 U/L. It indicated that all of the fragments had GGT enzyme activity, and the enzymic center was located in the C-terminal. The fusion expression was demonstrated by western-blot detecting the GFP fragment. Under microscopy observation, the products were mainly located in the cytoplasm of the cell. When the N-terminal 380aa was deleted, the location was changed and the products were collected together.(3) Cell apoptosis induced by the GGT fragmentsThe voltage of the cell mitochondrial membrane was changed induced with the ggt products by JC-1 staining assay. It indicated that the ggt products can induce the cell apoptosis by mitochondrial pathway. Apoptosis of the Sf21 cells was clearly observed only by inducing with the full length ggt fragment. Furthermore, the fragment fusing other gene in the C-terminal did not affect the apoptotic activity. (4) The growth Inhibition of the ggt products to stomach cancer cell BGC-823The MTT method was used to detect the growth of stomach cancer cells (BGC-823) by stimulation of the ggt products. Only the full length GGT was found to function in inhibition of the cell growth. However, its function acted in early stage of stimulation. Following the incubation time, the effect of the stimulation, the cell growth recovered. It indicated that growth inhibition of the GGT product acted in a short time.Above all, even if the ggt gene was predicted to contain multiple function domains by analysis of bioinformatics, the N terminal structure was very important to its function. The growth inhibition of the GGT product acted in a short time.