淡色库蚊转铁蛋白在毕赤酵母中的分泌表达及其抑菌活性的初步研究

发布时间：2018-01-10 20:25

本文关键词：淡色库蚊转铁蛋白在毕赤酵母中的分泌表达及其抑菌活性的初步研究　出处：《中国寄生虫学与寄生虫病杂志》2014年01期 　论文类型：期刊论文

【摘要】：目的利用毕赤酵母(Pichia pastoris)分泌表达具有生物活性的淡色库蚊(Culex pipiens pallens)转铁蛋白(Transferrin,Tsf),并初步研究其抑菌活性。方法 RT-PCR扩增淡色库蚊转铁蛋白的编码基因序列,并克隆至毕赤酵母组成型表达载体pGAPZα-A的α-factor信号肽序列下游,构建pGAPZα-A-Tsf重组分泌表达载体。重组载体经XhoⅠ和XbaⅠ双酶切和测序正确后,采用BlnⅠ线性化处理,电击转化毕赤酵母GS115感受态细胞,转化子经Zeocin抗性筛选和菌落PCR构建pGAPZα-A-Tsf/GS115工程菌。重组转铁蛋白表达上清采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析。表达上清经镍-氨三乙酸亲和层析柱(NiNTA)纯化后,磷酸缓冲液(PBS)稀释为1.000、0.500、0.250和0.125 mg/ml,采用琼脂糖扩散抑菌圈法进行抑菌实验。结果 RT-PCR结果显示,转铁蛋白基因片段大小为2 100 bp,与预期结果一致。pGAPZα-A-Tsf重组载体双酶切获得2 127 bp条带,与预期结果一致,测序结果表明其构建成功。菌落PCR结果显示,pGAPZα-A-Tsf/GS115工程菌构建成功。SDS-PAGE和Western blotting结果显示,重组转铁蛋白表达了相对分子质量(Mr)约为80 200的蛋白产物,与预期大小一致。Ni-NTA纯化后重组转铁蛋白的抑菌实验表明,添加1.000、0.500、0.250 mg/ml重组转铁蛋白的平板上均有明显的抑菌圈,最低抑菌浓度为0.250 mg/ml。结论淡色库蚊转铁蛋白可通过基因重组方式从毕赤酵母中分泌表达具有抑菌活性的转铁蛋白。
[Abstract]:The purpose of using Pichia yeast (Pichia pastoris) expression with biological activity of Culex pipiens pallens (Culex pipiens pallens) transferrin (Transferrin, Tsf), and study its antibacterial activity. Encoding gene sequence was amplified by RT-PCR transferrin of Culex pipiens pallens, alpha -factor downstream signal peptide sequence and cloned into Pichia pastoris expression vector pGAPZ alpha -A, alpha pGAPZ construct -A-Tsf recombinant expression vector. The recombinant vector by Xho I and Xba I double enzyme digestion and sequencing, the Bln of linear processing, electroporation of Pichia pastoris GS115 by normal cells, transformants by Zeocin resistance screening and colony PCR to construct pGAPZ alpha -A-Tsf/GS115. The recombinant engineering bacteria the expression of transferrin supernatant using twelve sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis (Western blotting). The supernatant by nickel ammonia acetic acid Pro three And column (NiNTA) after purification, phosphate buffer (PBS) diluted to 1.000,0.500,0.250 and 0.125 mg/ml by agar diffusion method antibacterial bacteriostatic ring experiment. Results the results of RT-PCR showed that the transferrin gene fragment size was 2100 BP, consistent with the expected results of.PGAPZ alpha -A-Tsf recombinant vector digested 2127 BP bands, and the expected results, the results of sequencing showed that the constructed colony. PCR results showed that the pGAPZ alpha -A-Tsf/GS115 engineering bacteria were successfully constructed..SDS-PAGE and Western blotting results showed that the recombinant expression of transferrin relative molecular weight (Mr) of about 80200 protein products, showed that the Bacteriostatic Experiment of transferrin was consistent with the expected size of purified recombinant.Ni-NTA, adding 1.000,0.500,0.250 tablet mg/ml recombinant transferrin have obvious bacteriostatic ring, minimum inhibitory concentration was 0.250 mg/ml. conclusion Culex pipiens pallens transferrin Transferrin can be secreted from Pichia pastoris by gene recombination.

【作者单位】：丽水学院医学院;杭州师范大学医学院;
【分类号】：R384.1;Q966
【正文快照】： 转铁蛋白(transferrin)是血浆中主要的含铁蛋白质,负责运载由消化管吸收的铁和由红细胞降解释放的铁,广泛存在于无脊椎动物和脊椎动物体内,不同种属和组织之间转铁蛋白的含量差别很大[1]。转铁蛋白作为参与机体生理基础代谢活动的铁离子转运蛋白,在结构上具有一定的保守性,其

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