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To construct prokaryotic expression plasmid of RPS13, express and purify the protein for the preparation of polyclonal antibody.RPS13 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the highly expressed cell of gastric multidrug cell SGC7901/VCR. After sequenced, the gene was cloned into the expression vector pET-28a(+) to construct RPS13 expression plasmid pET-28a(+)-RPS13. The expression plasmid was transformed into the E.coli BL21 and screened by ampiline, and then the E.coli BL21 containing the expression plasmid was induced by IPTG and the protein was purifed by Histag column. BALB/c mice were immunized by the immunogen His-RPS13. The titer was measured by ELISA and the polyclonal antibody was obtained.The expression plasmid pET-28a(+)-RPS13 was constructed. The 19 kDa recombined protein was successfully expressed in the E.coli BL21 by IPTG for 3 hours, purified by Histag column and confirmed by SDS-PAGE and Western blot. The polyclonal anti-His-RPS13 antibody was obtained by immunizing the mice with RPS13 protein. RPS13 could be specifically recognized by the antibody based on Western blot.RPS13 has bene expressed and purified successfully and polyclonal anti-His-RPS13 antibody has been prepared. Our study can be used for the preparation of monoclonal anti-His-RPS13 antibody and for further research of the function of the RPS13 protein in tumor.

To express an immunochemotactic and angiostatic anti-tumor molecule, CXC chemokine Crg-2, by E. coli recombinant system of adenovirus.By E. coli recombination system of adenovirus, the shuttle vector pShuttle-cmv/crg-2 was co-transfected into BJ5183 E. coli with adenovirus backbone plasmid pAdEasy-1. The recombinants were transfected into 293 packaging cells and adenovirus was packaged and amplified. The molecular weight and bioactivity of recombinant protein were determined by Western blot and chemotaxis assay, respectively.Recombinant adenovirus genome backbone bearing crg-2 gene, pAd/crg-2, was successfully obtained as evidenced by endonucleases digestion and PCR. Adenovirus was packaged and amplified to a titre of 4x10(9) TCID50/L. The cultured supernatant of the infected 293 cells contented a protein of nearly 10,000 and presented a significantly chemotatic effect towards activated spleen lymphoblasts.Recombinant adenovirus Ad/crg-2 obtained by E. coli recombinant system of adenovirus can efficiently express bioactive Crg-2 chemokine.

To construct the RNA interference eukaryotic expression vector specific for human MAD2 gene and to observe its effect on the growth of gastric cancer cell line SGC7901.The expression vectors of pSilencer3.1/MAD2-siRNA1 and pSilencer3.1/MAD2-siRNA2 were constructed by gene recombination and then were stably transfected into the gastric carcinoma cell line SGC7901 by liposome mediation. The expression of MAD2 on the levels of protein and mRNA was detected by Western blot and RT-PCR, and the monoclone with the highest inhibition efficiency was selected. The growth of the transfected cells was assessed by MTT. And the cells treated with 1.0 mg/L vincristine (VCR) for 24 h were analyzed by FCM for cell cycle.Sequence-specific siRNAs targeting MAD2 significantly down regulated the expression of MAD2 in SGC7901 cells. In MAD2-siRNA transfected cells, the rate of cell growth increased markedly and cell cycle couldn't be arrested in M phase induced by VCR, while the cells transfected with the mock vector could.Down regulation of MAD2 expression of SGC7901 bv sequence-specific siRNA could accelerate the cell growth and impair the mitosis arrest of SGC7901 induced by VCR.

To construct prokaryotic vector GX1-rmhTNF containing neovascular-targeting peptide GX1 and human TNFalpha and produce the GX1-rmhTNF protein.We have previously obtained a specific phage peptide CGNSNPKSC (GX1) binding to vasculature of human gastric cancer. The GX1-rmhTNFalpha vector was constructed by merging sequence of neovascular-targeting peptide GX1 with N-terminal of new recombined human TNFalpha (rmhTNFalpha) using gene engineering methods. The expression of the fusion protein GX1-rmhTNFalpha in E. coli was induced by temperature. The expression of GX1-rmhTNFalpha was detected by SDS-PAGE and Western blot.A novel protein with expected molecular mass about 18,000 was found after SDS-PAGE and gel staining. The expressed product showed a good binding ability to anti-TNFalpha monoclonal antibody.The prokaryotic vector GX1-rmhTNF was constructed and the expression of GX1-rmhTNF protein was successfully induced, which was helpful for further purification of GX1-rmhTNF protein.

To express hCacyBP in E.coli and prepare mouse polyclonal antibody against hCacyBP so as to study tissue distribution of hCacyBP and evaluate its role during formation of multidrug resistance to gastric cancer.hCacyBP gene was subcloned into an expression vector pET28a, and then the recombinant vector was transformed into E. coli BL21. The recombinant protein was expressed in BL21 under IPTG induction. Using purified hCacyBP as immunogen, mouse polyclonal antibody against hCacyBP was prepared. hCacyBP was detected by Western blot in 10 kinds of rabbit tissues. Expression and distribution of hCacyBP in SGC7901/VCR and SGC7901 cells were detected by Western blot and immunohistochemical staining.hCacyBP was successfully expressed in E. coli. The Western blot analysis showed that hCacyBP was expressed in all 10 kinds of rabbit tissues, but expression of brain and liver tissues were higher as compared with other tissues. Expression and distribution of hCacyBP in both SGC7901/VCR and SGC7901 cells had no significant difference.CacyBP expressed widespreadly in varied tissues. Polyclonal antibody against hCacyBP that we prepared has high specificity, which provides a powerful tool for studying the function of hCacyBP.

To study the expression of RhoC in human gastric cancer cell lines and to construct and identify the RhoC-specific siRNA expressing vector.The expression of RhoC in human gastric cancer cell lines was detected by Western blot. According to the computer aided design(CAD),RhoC-specific siRNA gene was synthesized and cloned into the expression vector mU6pro. The constructed RhoC-siRNA was transiently transfected into high-metastatic gastric cancer cell line AGS and the inhibition effect of RhoC-siRNA on expression of RhoC in AGS cells was detected using Western blot.The expression level of RhoC was up-regulated in high metastatic cells lines. Double enzyme digestion analysis and DNA sequencing confirmed that the RhoC-specific siRNA expression vector was constructed successfully. RhoC expression in AGS cells was specifically suppressed after transfection of RhoC-siRNA.The RhoC-specific siRNA expression vector has been successfully constructed, which may provide a novel applicable strategy for gene therapy of gastric cancer.

To screen in vivo from a phage-displayed peptide library polypeptide fragments specific binding to vascular endothelial cells of gastric cancer xenografts, so as to provide for anti-angiogenesis therapy of tumor.Immunosupressed mice models for human gastric cancer xeno-grafts were established by subrenal capsular assay (SR-CA). The 12-peptide library was panned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue (brain). The distribution of phage were detected in transplanted tumor tissues by immunohistochemical staining.Phage homing to gastric cancer xenografts were enriched through four rounds of panning,being 3.4-fold of that recovered from brain tissue. Peptide sequences were characterized for randomly picked-upclones and the peptide sequence YESIRIGVAPSQ appeared most frequently. Immunohistochemical staining for the homing phage revealed a specific vascular endothelial cell localization in gastric cancer xenografts 5 min after injection of the enriched phages. When the specific phage individually test-ed, the phage recovered from gastric cancer xenografts were as 4. 2 times as those from control tissue ( brain) , as 4.9 times as those from lung, as 5.4 times as those from heart.The tumor-specific homing peptides may provide a effective tool for targeting tumor vasculature in anti-angiogenesis therapy of cancer. The in vivo selection technique in this study was feasible and applicable to screening peptides homing to vascular endothelial cells.

To screen bioactive peptides that mimic the epitope of gastric cancer associated antigen.Anti-gastric cancer monoclonal antibody (mAb) MG7 was purified by ion chromatography, and then coated on ELISA plate. By using the mAb as selective molecular, a 12-meres phage displaying peptide library was biopanned, and the positive clones were selected.12 positive phage clones were selected.A candidate mimic epitope of gastric cancer associated antigen was identified, which provided the foundation for developing tumor peptide vaccine.

To clone and express the testicular carcinoma antigen MAGE-E1 gene in E.coli.The cDNA encoding human MAGE-E1 gene was amplified by RT-PCR from human glioma cell line BT-325, then the MAGE-E1 gene was inserted into plasmid pGEM-T easy. After sequencing, the MAGE-E1 was cloned into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant expression vector pGEX-4T-2-MAGE-E1 which was used to transform E.coli.The expressed product reached the highest level at 5 h after IPTG induction. SDS-PAGE and scanning analysis of gel density indicated that the expressed protein was about M(r) 41 000 and account for 35% of the total bacterial protein.The high efficient expression of the MAGE-E1 gene fragment lays the foundation for further preparing antibody against MAGE-E1 protein and the tumor vaccine for glioma immunotherapy.