抗菌肽Alloferon-1串联式重组表达及其表达产物体外抗肿瘤活性的研究

Serial recombinant expression and activity against tumor cells in vitro of antibacterial peptide Alloferon-1

目的:构建串联式表达Alloferon-1的原核重组表达系统,检测合成及重组表达的Alloferon-1体外抗肿瘤细胞活性。方法:采用连接引物PCR法,构建含6×His-EK-8×Alloferon-1-EK-6×His序列的人工融合基因;采用常规分子生物学方法克隆并构建该人工融合基因及其原核表达系统;采用SDS-PAGE和BioRad凝胶图象分析系统了解目的重组产物8×rAlloferon-1-EK表达情况和产量;采用Ni-NTA亲和层析及EK酶切和Sephadex G-50层析法提纯8×rAlloferon-1-EK及rAlloferon-1-EK;采用MTT法检测rAlloferon-1-EK体外抑制KB、SGC和HL-60肿瘤细胞增殖的作用,并与直接合成的Alloferon-1(sAlloferon-1)和Alloferon-1-EK(sAlloferon-1-EK)的抗肿瘤作用比较。结果:获得了序列正确的目的人工融合基因克隆及其原核表达系统pET42a-8×rAlloferon-1-EK-E.coliBLDE3。在IPTG诱导下,该原核重组表达系统可表达串联式目的重组蛋白8×rAlloferon-1-EK,其产量约为细菌总蛋白的30%。Ni-NTA和Sephadex G-50层析后,可分别获得8×rAlloferon-1-EK和rAlloferon-1-EK。在25~100μg/ml剂量范围内,sAlloferon-1、sAlloferon-1-EK和rAlloferon-1、rAlloferon-1-EK均有明显抑制KB、SGC和HL-60肿瘤细胞生长及增殖的效果(P<0.01),且四者抑制作用无明显差异(P>0.05)。结论:本研究成功构建了串联表达rAlloferon-1的原核表达系统,其表达产物具有与合成的Alloferon-1和Alloferon-1-EK相似的体外抗肿瘤细胞活性。

Objective： To construct a prokaryotic expression system to serially express Alloferon-1 and to determine the anti-tumor activity of its products in vitro. Methods： An artificial fusion gene containing 6 × His-EK-8 × Alloferon-1-EK-6 × 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 × rAlloferon-1-EK. Ni-NTA affinity chromatography and EK digestion and Sephadex G-50 chromatography were performed to extract 8 × 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. Results： The target artificial fusion gene and its prokaryotic expression system pET42a-8 × 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 × rAlloferon-1-EK and its output was approximate 30% of the total bacterial proteins. 8 × rAlloferon-1-EK and rAlloferon-1-EK were obtained through Ni-NTA and Sephadex G-50 columns, sAlloferon-1, sAlloferon-1-EK and rAlloferon-1, rAlloferon-EK showed similar remarkable effects of inhibiting the growth and proliferation of KB,SGC and HL-60 cells in vitro within 25- 100 μg/ml concentration range （P〈 0.01） ,and there were no significant differences in the inhibiting effects among the three agents （P^0. 05）. Conclusions： 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.