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| Serial recombinant expression and anti-tumor activity in vitro of antibiotic peptide Alloferon-1 |
| XU Xiao-yin 1,YAN Jie 1,SUN Qi 2 |
| 1.Department of Medical Microbiology and Parasitology,Zhejiang University School of Medicine,Hangzhou 310058,China; 2.Department of Laboratory Medicine,Zhejiang Medical College,Hangzhou 310053,China |
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Abstract Objective： To generate a recombinant expression system of repeated serial antibiotic peptide Alloferon-1 DNA segment with trypsin digestion site and to determine its anti-tumor activity in vitro. Methods： A 14 repeated serial DNA segment of Alloferon-1 with a lysine residual at the C-end that acts as the trypsin digestion site was constructed.pET42a vector and E.coli BL21DE3 were applied to generate the prokaryotic expression system of the repeated serial DNA segment of Alloferon-1.The yield of target recombinant product was measured by SDS-PAGE and Bio-Rad Gel image system.Ni-NTA affinity column,trypsin digestion and Sephadex G-50 column were used to purify 14 rAlloferon-1-K fusion protein and rAlloferon-1-K monomer.By using the co-cultivation of BALB/c mouse splenocyte with K562,KB or SGC tumor cells and CCK-8 detection method,the effects of rAlloferon-1-K,chemosynthetic Alloferon-1 (cAlloferon-1) and Alloferon-1-K (cAlloferon-1-K) on the growth and proliferation of tumor cells were detected. Results： The prokaryotic expression system E.coli BL21DE3pET42a-14 Alloferon-1-K efficiently expressed 14 rAlloferon-1-K fusion protein under inducement of IPTG,and the yield of fusion protein was approximate 30% of the total bacterial proteins.0.1~10 ng/ml rAlloferon-1-K remarkably increased the effect of mouse splenocytes to inhibit the growth and proliferation of K562,KB and SGC cells (P<0.05),and there was no statistically significant difference of the anti-tumor ability of rAlloferon-1-K compared to that of cAlloferon-1 or cAlloferon-1-K (P>0.05). Conclusion： A prokaryotic expression system of repeated serial Alloferon-1 DNA segment has been successfully constructed with high yield of rAlloferon-1-K,which maintains anti-tumor activity in vitro.
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Published: 25 September 2011
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Alloferon-1抗生肽串联重组表达及重组Alloferon-1体外抗肿瘤活性
目的：构建含胰蛋白酶酶切位点的Alloferon-1抗生肽重复串联DNA片段及其原核表达系统，了解有赖氨酸尾重组Alloferon-1（rAlloferon-1）体外抗肿瘤活性。 方法：根据Alloferon-1序列不含精氨酸和赖氨酸的特点，构建C端加有含胰蛋白酶酶切位点赖氨酸的14×Alloferon-1抗生肽重复串联DNA片段。采用pET42a表达载体和E.coli BL21DE3表达宿主菌，构建Alloferon-1重复串联DNA片段原核表达系统。SDS-PAGE联合Bio-Rad凝胶图像分析系统检测目的重组产物表达量，Ni-NTA亲和层析、胰蛋白酶酶切和Sephadex G-50层析法提纯14×rAlloferon-1-K及其rAlloferon-1-K单体。BALB/c 小鼠脾细胞分别与K562、KB和SGC肿瘤细胞共培养模，采用CCK-8法检测rAlloferon-1-K、化学合成Alloferon-1 （cAlloferon-1）及Alloferon-1-K（cAlloferon-1-K）体外抑制肿瘤细胞生长及增殖的活性并进行比较。 结果：原核表达系统E.coli BL21DE3pET42a-14×Alloferon-1-K在IPTG诱导下能有效表达14×rAlloferon-1-K蛋白，其产量约为细菌总蛋白的30%。0.1～10 ng/ml的rAlloferon-1-K具有明显提高小鼠脾细胞抑制K562、KB和SGC肿瘤细胞的生长及增殖（P<0.01），rAlloferon-1-K的抗肿瘤活性与cAlloferon-1及cAlloferon-1-K比较，差异无统计学意义（P>0.05）。 结论：本研究成功构建了Alloferon-1抗生肽重复串联原核表达系统，有效地提高了Alloferon-1产量；而且rAlloferon-1-K仍保持原有的体外抗肿瘤活性。
关键词：
重组融合蛋白质类,
肽类/分离和提纯,
抗菌药/分离和提纯,
抗肿瘤药/药理学,
重组,
遗传,
质粒
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