Guanylate-binding protein
a technology of guanylate and binding protein, which is applied in the field of guanylate binding protein, can solve the problems that stomach cancer is a leading cause of cancer deaths worldwid
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[0205] Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Rockville, Md.
example i
Cloning of Human GBP-4
1. Materials and Methods
Cells and Reagents
[0206] The following cell lines were obtained from the American Type Culture Collection (Rockville, Md.) and grown as recommended: 293, human embryonic kidney cells, HeLa, human epitheloid carcinoma of the cervix (ATCC, CCL-2), A431, human epidermoid carcinoma (ATCC, CRL-1555), RF-1, derived from a human primary gastric adenocarcinoma (ATCC, CRL-1864), RF-48, derived from a human gastric adenocarcinoma metastatic to peritoneal fluid (ATCC, CRL-1863), AGS, derived from a human primary gastric adenocarcinoma (ATCC, CRL-1739), KATO III, a human gastric carcinoma metastatic to pleural fluid (ATCC, HTB-103), and Hs746T, derived from a human stomach carcinoma metastatic to the left leg (ATCC, HBT-135). Purified recombinant human IFN-γ was obtained from Genentech, Inc. The rHuIFN-γ and rHuIFN-α used in this example displayed specific anti-viral activities of 2×107 and 5×106 IU / mg, respectively (1.0 IU of IFN-α per ml=30 f...
example ii
E. coli Expression of cDNA Encoding Human GBP-4
[0253] An expression vector from Qiagen Inc. called pQE-30 was used for this example. A restriction map of pQE-30 is shown in FIG. 8. The plasmid for GBP-4 of Example I was digested with BamHI and SalI. The resulting DNA fragment was ligated into pQE-30 previously cut with BamHI and SalI to accommodate the DNA fragment using standard ligation methodology as described in Sections 5.10 to 5.11 of Sambrook et al., supra. The resulting vector is called pQE30.hu.GBP4.
[0254] Competent E. coli 27C7 cells were transformed with pQE30.hu.GBP4 by standard transformation techniques. Transformants were selected and purified on LB plates containing 20 mg / L tetracycline. This medium had the following composition: 10 g / L Bacto-Tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 mg / L tetracycline-HCl.
[0255] One transformed colony was used to inoculate sterile LB broth containing 20 mg / L tetracycline. The flask culture was incubated at 35-39...
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