[Serial recombinant expression and activity against tumor cells in vitro of antibacterial peptide Alloferon-1].
To construct a prokaryotic expression system to serially express Alloferon-1 and to determine the anti-tumor activity of its products in vitro.



An artificial fusion gene containing 6 x His-EK-8 x Alloferon-1-EK-6 x His sequences was constructed by linking primer PCR. By using routine molecular biological methods, the artificial fusion gene was cloned and its prokaryotic expression system was then constructed. SDS-PAGE and BioRad Agarose Image Analysor was applied to measure the expression and output of the target recombinant products 8 x rAlloferon-1-EK. Ni-NTA affinity chromatography and EK digestion and Sephadex G-50 chromatography were performed to extract 8 x rAlloferon-1-EK and rAlloferon-1-EK, respectively. The proliferation of KB, SGC and HL-60 tumor cells was tested by using MTT method after treatment with directly synthesized Alloferon-1 (sAlloferon-1), Aloferon-1-EK (sAlloferon-1-EK) and rAlloferon-1-EK.



The target artificial fusion gene and its prokaryotic expression system pET42a-8 x rAlloferon-1-EK-E. coliBLDE3 with the expected sequences were obtained. Under inducement of IPTG, the prokaryotic expression system expressed the target serial recombinant protein 8 x rAlloferon-1-EK and its output was approximate 30 % of the total bacterial proteins. 8 x rAlloferon-1-EK and rAlloferon-1-EK were obtained through Ni-NTA and Sephadex G-50 columns. sAlloferon-1, sAlloferon-1-EK and rAlloferon-1ìrAlloferon-EK showed similar remarkable effects of inhibiting the growth and proliferation of KB, SGC and HL-60 cells in vitro within 25 approximately 100 microg/ml concentration range (P<0.01), and there were no significant differences in the inhibiting effects among the three agents (P>0.05).



A prokaryotic expression system to serially express rAlloferon-1 has been successfully constructed. The product rAlloferon-1-EK has a similar anti-tumor activity compared to both the synthesized Alloferon-1 and Alloferon-1-EK in vitro.
Sun
Qi
Q
Zhejiang Medical College, Hangzhou 310053, China.
Sun
Ai-hua
AH
Yan
Jie
J
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006131:Growth Inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D018922:HL-60 Cells; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007624:KB Cells; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
C510535:Alloferon; D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D001428:Bacterial Vaccines; D006131:Growth Inhibitors; D010455:Peptides; D011993:Recombinant Fusion Proteins