抗菌肽Alloferon-1串联式重组表达及其产物体外抗病毒活性的研究

Serial recombinant expression of antibacterial peptide alloferon-1 and antivirus activity of product in vitro

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体外抗病毒活性。方法:采用基因工程技术构建含6×His-EK-8×Alloferon-1-EK-6×His序列的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CPE观察和MTT法检测rAlloferon-1-EK体外抗IV-A3、CV-B4和RSV-E6株的作用,并与直接合成的Alloferon-1(sAllofer-on-1)和Alloferon-1-EK(sAlloferon-1-EK)的抗病毒活性比较。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。该原核重组表达系统串联式表达的目的重组蛋白8×rAlloferon-1-EK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。25μg/ml的各Alloferon-1s均无明显的抗病毒作用,50μg/ml和100μg/ml的各Alloferon-1s有抗IV-A3和RSV-E6株作用且活性相似(P>0.05),但各Alloferon-1s均无抗CV-B4株的作用。结论:本研究成功地构建了串联表达Alloferon-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相似的体外抗病毒活性。

Objective ：To construct a prokaryotic expression system to serially express Alloferon - 1 and determine the activity against viruses in vitro of synthesized and recombinant expressed Alloferon - 1 s. Methods：An artificial fusion gene containing 6 × His - EK - 8 × Alloferon - 1 - EK - 6 × His sequences and its prokaryotic expression system were constructed by genetic engineering technique. SDS - PAGE plus Bio - Rad Agarose Image Analyzor was applied to measure the expression and output of the target recombinant product 8 × rAlloferon - 1 - EK. Ni - NTA affinity chromatography and EK digestion plus Sephadex G - 50 chromatography were performed to extract 8 x rAlloferon - 1 - EK and rAlloferon - 1 - EK, respectively. By using CPE index and MTT, the antivirus effects to IV- A3, CV- B4 and RSV- E6 in vitro of rAlloferon- 1 -EK, synthesized Alloferon- 1 （sAlloferon- 1 ） and Aloferon - 1 - EK （ sAlloferon - 1 - EK） were detected as well as compared. Results：The target artificial fusion gene and its prokaryotic expression system pET42a - 8 × rAlloferon - 1 - EK - E. coli BLDE3 with the expected sequences were established in this study. Output of the target serial recombinant protein 8 x rAlloferon - 1 - EK expressed by the prokaryotic expression system was approximate 30% of the total bacterial proteins. After going Ni - NTA and Sephadex G - 50 columns, 8 × rAlloferon - 1 - EK and rAlloferon - 1 - EK were respectively obtained. 25 μg/ml of each the Alloferon - Is did not present significant antivirus activity to all the three viruses. 50 μg/ml or 100 μg/ml of each the Alloferon-1s showed remarkable activities against IV -A3 and RSV - E6 and the antivirus effects were similar （ P 〉 0. 05 ）. However, all the Alloferon - 1 s had no antivirus ability to CV - 134. Conclusion： A prokaryotie expression system to serially express rAlloferon - 1 is successfully constructed in this study. rAlloferon - 1 - EK has a similar antivirus activity compared to both the synthesized Alloferon - 1 and Alloferon - 1 - EK in vitro.