Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.
The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.
Department of Rheumatology, the First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, 710048, People's Republic of China.
Sheng-Juan
SJ
Hu
Rong-Xing
RX
Jiang
Hua-Hong
HH
Xie
Ai-Ling
AL
Ma
Hong-Li
HL
Shi
Hao
H
Shen
Zhi-Ming
ZM
Hao
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000517:prevention & control* / Q000556:secondary*; D051379:Mice; D009361:Neoplasm Invasiveness; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000627:therapeutic use; D013274:Stomach Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D010455:Peptides; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha