Cloning And Expression Of Gastric Cancer Related Gene (GCRG213, GCRG224) In E.coli As Well As Preparation Of Polyclonal Antibody

A differentially expressed gene, which was named W2, was screened directly by fluorescent differential display reverse transcription polymerase chain reaction (DDTR-PCR) analysis from human intestinal-type gastric cancer, paratumor and non-tumor tissues in our laboratory, and the results showed its expression much higher in all seven tested tumor and paratumor samples than in their normal counterparts, it was sub-cloned into a pGEM-T Easy vector. Two subclones, GCRG213 (gastric cancer related gene 213) and GCRG224, were identified from the subcloning procedure.GCRG224 consisted of 1159 base pairs and had one open reading frame (ORF). The GCRG224-ORF consists of 35 amino acids with an estimated molecular weight of 3.8 kDa. It located at human chromosome Ilql4. No homologue was found in GenBank database with GCRG224-ORF. This nucleotide sequence data was submitted to GenBank with accession No. AF438406. In situ hybridization analysis showed that only 5/30 adenocarcinoma, 3/18 dysplasia and 6/18 intestinal metaplasia showed higherGCRG224 expression level than the normal gastric glands. However, GCRG224 was over-expressed predominantly in 26/30 cases of normal mucosal epithelium.GCRG213 consisted of 1094 base pairs with an open reading frame (ORF) which encodes 142 amino acids with an estimated molecular weight of 16.4 kDa. This nucleotide sequence data was submitted to GenBank with accession No. AY053451. Through conserved domain database search in GenBank, a putative conserved domain, Human apurinic (apyrimidinic) endonucleas/redox-factor 1 (APE/Ref-1), was detected in the deduced amino acid sequence of GCRG213-ORF, it shared 61.0% alignment with the C-terminal region of APE/Ref-1 conserved domain. APE/Ref-1 is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. It is the major AP endo activity in human cells and is a key enzyme in base excision repair pathways that remove alkylation-induced abasic sites. APE/Ref-1 associates with p53 and is a potent activator of p53 binding in vivo, enhancing the ability of p53 to transactivate a number of p53 target promoters. Down-regulation of APE/Ref-1 causes a reduction in the ability of p53 to transactivate the p53 and Bax promoters. Thus, APE/Ref-1 not only plays a key role in BER, but also is a key regulator of p53 and, hence, of cell cycle arrest and apoptosis.APE/Ref-1 levels have been found to be elevated in a number of cancer such as ovarian, cervical, prostate, colorectal and germ cell tumors, malignant gliomas, and in breast cancer, APE protein expression correlates with lymph node status and angiogenesis. In10our study, GCRG213 overexpressed in early and advanced gastric adenocarcinoma. The patterns of GCRG213 expression in cancerous tissue of the early gastric cancer did not differ significantly from the advanced gastric carcinoma. This expression pattern is in consistent with that of APE in cervical, prostate, colorectal cancer and their premalignant leisions reported. Because of the similarity of the expression pattern in tumors between APE and GCRG213, as well as the 61% alignment between the amino acid sequences of GCRG213-ORF and APE conserved domain, it is likely that GCRG213-ORF was a new member of the APE family.As such, it may play a important role in the diagnosis and therapy of gastric cancer to clarity the role of GCRG213 and GCRG224 in the development of gastric carcinoma.In order to further study the the biological function of GCRG213 and GCRG224 in the development of gastric carcinoma and the value in the diagnosis and therapy of gastric cancer, we conducted following experiments: GCRG213 and GCRG224 were expressed in E.coli to obtain purified GCRG213 and GCRG224 recombinant proteins, and prepared the polyclonal antibodies. The experiment procedures and results are as follows:1. GCRG213 and GCRG224 cDNA with complete open reading frame were amplified by PCR from plasmid pGEM-T, and then were cloned into prokaryotic expression vector pET102/D-TOPO to obtain two positive re...