發(fā)布時(shí)間：2018-11-18 21:34

【摘要】：目的;利用基因工程的方法,在畢赤酵母菌(Pichia patoris,P.pastoris)中成功表達(dá)Apidaecin型抗菌肽。方法:利用PCR技術(shù)在Apidaecin基因N端插入EcoR I酶切位點(diǎn)、GST標(biāo)簽(并連接DDDDK腸激酶位點(diǎn)),且在C端插入終止密碼子和Not I酶切位點(diǎn),后將上述片段擴(kuò)增后與表達(dá)載體pPICZαA連接,構(gòu)建重組表達(dá)載體pPICZαA-GST-Apidaecin,并測(cè)序鑒定;將pPICZαA-GST-Apidaecin重組質(zhì)粒電轉(zhuǎn)至P.pastoris X33中并進(jìn)行發(fā)酵表達(dá);表達(dá)產(chǎn)物經(jīng)GST親和層析純化后進(jìn)行SDS-PAGE凝膠電泳鑒定,EK腸激酶切除GST標(biāo)簽后進(jìn)行抑菌實(shí)驗(yàn)。結(jié)果:SDS-PAGE凝膠電泳結(jié)果顯示GST-Apidaecin融合蛋白表達(dá)的產(chǎn)物條帶位于分子量約為28KD處,與理論分子量一致;抑菌實(shí)驗(yàn)表明,用EK腸激酶切除GST融合標(biāo)簽后,Apidaecin型抗菌肽對(duì)大腸桿菌有明顯的抑菌活性。結(jié)論:Apidaecin型抗菌肽在P.pastoris菌中得到成功表達(dá)并具有抑菌活性。
[Abstract]:Objective: to express Apidaecin type antimicrobial peptides successfully in Pichia pastoris (Pichia patoris,P.pastoris) by genetic engineering. Methods: PCR technique was used to insert EcoR I restriction site at the N end of Apidaecin gene, GST tag (and ligation of DDDDK enterokinase site), and terminal codon and Not I restriction site to be inserted at C terminal. The above fragments were amplified and ligated with the expression vector pPICZ 偽 A. The recombinant expression vector pPICZ 偽 A-GST-Apidaecinwas constructed and sequenced. The recombinant plasmid of pPICZ 偽 A-GST-Apidaecin was transformed into P.pastoris X33 and expressed by fermentation, and the expressed product was purified by GST affinity chromatography and identified by SDS-PAGE gel electrophoresis. EK enterokinase was removed from GST label for bacteriostasis test. Results: the results of SDS-PAGE gel electrophoresis showed that the product bands expressed by GST-Apidaecin fusion protein were located at the molecular weight of 28KD, which was consistent with the theoretical molecular weight. The bacteriostatic test showed that Apidaecin antibacterial peptide had obvious bacteriostatic activity against Escherichia coli after GST fusion label was removed by EK enterokinase. Conclusion: Apidaecin type antimicrobial peptide was successfully expressed in P.pastoris strain and had bacteriostatic activity.
【作者單位】： 蘭州大學(xué)口腔醫(yī)學(xué)院;西北民族大學(xué)甘肅省口腔疾病研究重點(diǎn)實(shí)驗(yàn)室培育基地西北民族大學(xué)口腔醫(yī)學(xué)國(guó)家民委重點(diǎn)實(shí)驗(yàn)室西北民族大學(xué)口腔醫(yī)學(xué)院;蘭州大學(xué)附屬第一醫(yī)院;隴南市第一人民醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào):31360124,31560159)
【分類號(hào)】：Q78

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