【作者】 侯瑞；

【导师】 胡福泉；

【作者基本信息】 第三军医大学 ， 微生物学， 2007， 硕士

【摘要】 研究背景:肽抗生素(peptide antibiotics)是生物体基因编码的、具有抗微生物活性的小肽。它通常由12～60个氨基酸所组成,分子量小于10kD,是生物体天然免疫的重要组成部分,是宿主在感染之后、机体的特异性免疫建立之前,控制微生物生长的重要物质。与传统的抗生素相比,它们具有广谱、高效的杀菌活性。在临床上耐药性问题日益棘手的今天,肽抗生素的研究和开发得到广泛重视。本室曾用简并引物从人皮肤角质形成细胞中克隆到一人β型肽抗生素,称为hPAB-β,并通过化学合成该肽证实了其杀菌活性。在原核表达系统中,曾以融合蛋白的形式表达该肽,但实际表达量占菌体总蛋白的比率仅约7%;也设计过多拷贝串联体来提高目标肽的产量,通过制备工艺的优化,在10升发酵罐中高密度发酵hPAB-β(3)/M15目的肽的表达率可达30.2%。但通过原核表达系统制备肽抗生素hPAB-β仍存在一些问题,如经过化学裂解、纯化、复性等处理,能获得活性目标肽,但回收率较低(约20%),使得最终复性得到的目标肽的产量较低,不利于规模化制备。因此寻找更好的表达系统,提高目标肽的表达量,得到回收率及纯度较高的产物是hPAB-β高效制备及临床前研究必须解决的问题。为此,我们选择了毕赤酵母表达系统来表达hPAB-β,展开了本课题的研究。研究内容与结果:1. hPAB-β毕赤酵母表达载体的构建:设计构建两类重组质粒。①含有6×His纯化标签:A:含天然hPAB-β序列的重组pPIC9K质粒。B:根据毕赤酵母密码子偏嗜性,构建含优化hPAB-β序列的重组pPIC9K质粒。②不含6×His纯化标签:含优化hPAB-β序列的重组pPIC9K质粒。我们首先用PCR及引物延伸法得到含标签的天然及优化的hPAB-β序列,经SnaB I和Not I双酶切后,与经过同样双酶切的pPIC9K质粒连接,转化DH5α大肠埃希菌,提取重组质粒,用PCR、双酶切和核苷酸测序鉴定。对于无标签的优化hPAB-β序列,我们以含标签的优化hPAB-β基因为模板,设计PCR引物扩增目的基因序列,经SnaB I和Not I双酶切后,与经过同样双酶切的pPIC9K质粒连接,转化DH5α大肠埃希菌,同样用PCR、双酶切和核苷酸测序鉴定。结果表明,从这两类重组质粒中可分别扩增出约167bp和144bp大小的片段,经Xho I/Not I双酶切也可切出约167bp和144bp大小的片段,与预期结果相符。核苷酸测序证实,含标签的天然hPAB-β序列有一同义突变,阅读框正确。含标签和无标签优化的hPAB-β序列和阅读框均完全正确。2. hPAB-β重组毕赤酵母的构建:将构建好的两类重组质粒经Sal I酶切线性化后,电转化整合入GS115毕赤酵母中,用浓度逐渐增加的G418来筛选多拷贝菌株,并鉴定其表型。对筛选出的菌株在DNA、mRNA及蛋白水平进行鉴定。结果表明,通过提取转化子的基因组进行PCR扩增,可得到约167bp和144bp大小的片段;经过对转化子的诱导表达,RT-PCR可扩增出约167bp和144bp大小的片段,均与预期结果一致。3.摇瓶水平表达目标肽的纯化与活性鉴定:对于含有6×His标签的目标肽先用Ni-NTA亲和层析进行初步纯化,再用P2分子筛层析脱盐进一步纯化,可得到较纯的目标肽;对于不含6×His标签的目标肽则用Source-30 RPC柱进行反相层析初步纯化。纯化后Tricine-SDS-PAGE分析显示,在约5.6kD和4.5kD处检测到目的蛋白条带。经抑菌实验验证,纯化后的两类重组目标肽对金黄色葡萄球菌和绿脓杆菌临床分离株具有良好的抑菌作用。4. hPAB-β工程菌高密度发酵条件的研究:与摇瓶水平表达相比,大规模高密度发酵时工程菌的生长环境不同,受培养基成分、细胞密度、溶氧值等诸多参数的影响。因此,在初步确定hPAB-β的表达条件后,用发酵罐深入探讨毕赤酵母高密度发酵表达无标签hPAB-β的条件:采用补料分批发酵的方式,接种工程菌于BMGY培养基中培养,作为一级种子菌;再在200ml BMGY培养基中按1%的比例接种一级种子菌作为二级种子菌。然后将二级种子菌按10%的比例接种于2L基础盐培养基的发酵罐中,同时添加微量元素培养基,通过调节转速将溶氧控制在70%以上,pH值控制在(5±0.05),温度控制在30℃,在初速度600r/min的条件下密集生长。待培养基中甘油耗竭,溶氧开始上升时添加补料生长培养基,每小时10ml/L,直到菌体湿重达到200～250g/L。停止流加补料生长培养基3 h,然后向罐中流加补料诱导培养基,开始用甲醇诱导菌体表达,每小时3ml/L持续3 h,之后增加到每小时9ml/L,在此期间pH值仍控制在5.0左右。按此条件,发酵上清经Tricine-SDS-PAGE分析可以看到有分子量约4.5kD的蛋白表达,与预期结果相符。三次发酵上清的总蛋白浓度分别为664.0μg/ml、781.8μg/ml和721.3μg/ml,目标肽的表达率达分别为11.1%、9.8%和10.3%。那么,三次发酵上清中目标肽的表达量分别为73.7mg/L、76.6 mg/L和74.3mg/L。5.高密度发酵表达hPAB-β的纯化与活性鉴定:对工程菌优5’高密度发酵表达无标签的hPAB-β后,将上清首先用Source-30 RPC柱进行反相层析初步纯化,再用P10分子筛层析分离不同大小的蛋白,同时达到脱盐的效果,Tricine-SDS-PAGE分析表明目标肽存在于P10分子筛的2峰中。之后将P10分子筛的2峰用Hi-pore Reversed Phase column进行反相高效液相色谱纯化,使目标肽纯度达95%以上。经抑菌实验验证,纯化后无标签hPAB-β对金黄色葡萄球菌临床分离株(MRSA)具有抑菌作用,且它对金黄色葡萄球菌临床分离株(MRSA)的抗菌活性强于新青Ⅱ、克林霉素,而略弱于去甲万古霉素。结论:本研究成功获得含标签的天然及优化的hPAB-β序列和无标签的优化的hPAB-β序列,在毕赤酵母表达载体pPIC9K基础上构建了两类重组质粒,筛选到能成功表达目标肽的高拷贝阳性转化子。在摇瓶表达的基础上进一步摸索了工程菌高密度发酵的条件,经过对重组目标肽的一系列纯化,得到了具有高纯度、较强生物学活性的hPAB-β,探索了一条更好的规模化制备重组hPAB-β的路线。更多还原

【Abstract】 Research background:Peptide antibiotics are small peptides encoded by organism genomic DNA. They are usually composed of 12～60 residues and are less than 10 kD. These peptides have been recognized to play important roles in the innate host defense in most living organisms. They have shown significance in controlling microorganisms before the hosts esteblish specific immunity after infected. Compared with conventional antibiotics, they have broad antimicrobial spectrums and high-performance antimicrobial activity. With the growing problem of resistance to conventional antibiotics, it has become important to study and develop peptide antibiotics.In our previous study, hPAB-βgene which encodes a beta peptide antibiotic was isolated from human keratinocytes in our laboratory, and it’s antimicrobial activity was confirmed by chemosynthesis hPAB-β. In prokaryotic expression system, the fusion protein was expressed in Escherichia coli JM109, and the actual expression level of the interest peptide was less than 7%. More over, tandem multimers were designed in order to improve the procduction of the active hPAB-β, and the expression level of the interest peptide coule reach 30% after reoptimization of the production artwork.But there are still some problems in using prokaryotic expression system to produce hPAB-β. For example, through chemical cleavage, purification and renaturation, active hPAB-βcan be obtained, but the recovery rate was very low, approximately at 20%. This is not benefit for us to obtain hPAB-βby prokaryotic expression system in large-scale. So, it is necessary to find a better expression system that can immprove the expression level and obtain purier target peptide. For these considerations, the Pichia pastoris expression system was chosen to get a high expression level of hPAB-β. Research contents and results:Firstly, constructed the Pichia pastoris expression vector. Two kinds recombinant plasmids were constructed.①With 6×His tag: A: Construct recombinant pPIC9K containing natural hPAB-βsequence. B: Accoding to the code in Pichia pastoris, construct recombinant pPIC9K containing optimised hPAB-βsequence.②Without 6×His tag: Construct recombinant pPIC9K containing optimised hPAB-βsequenc.We obtained natural and optimized sequence of hPAB-βwith tag by PCR and primer extension. After digested with SnaB I and Not I, the target sequences were ligated with pPIC9K which was also digested with the same enzymes. Then, transformed the recombinant plasmids into DH5α, isolated the recombinant plasmids, and identified them by PCR, enzyme digestion and nucleotide sequencing. We obtained optimised sequence of hPAB-βwithout tag by PCR, taking optimized sequence of hPAB-βwith tag as template. After digested with SnaB I and Not I, the target sequences were ligated with pPIC9K which was also digested with the same enzymes. Then, transformed the recombinant plasmid into DH5α, isolated the recombinant plasmid, and identified it still by PCR, enzyme digestion and nucleotide sequencing.We could amplify 167bp and 144bp fragments by PCR, and the same fragments could also be released from the recombinant plasmids as expected. Nucleotide sequencing showed that the natural sequence of hPAB-βhad a samesense mutation, and the open reading frames was confirmed to be right. While the optimised sequences of hPAB-βwith/without tag and their open reading frames were both confirmed to be all right.Secondly, constructed recombinant Pichia pastoris of hPAB-β. Digested the two kinds of recombinant plasmids with Sal I, then electrotransformated them into GS115. Screened multicopy transformants by G418, and identified their phaenotypes. Then identified the transformants in levels of DNA, mRNA and protein. The results showed that 167bp and 144bp fragments coule be amplified by PCR. After inducing the transformants, 167bp and 144bp fragments could be amplified by RT-PCR.Thirdly, purification and biological activity identification of target peptides expressed in level of flask. For hPAB-βwith tag, Ni-NAT affinity chromatography was first conducted. Then P2 gel filtration chromatography was subsequently used. After that, we could obtain peptide with less impurity. For hPAB-βwithout tag, reverse phase chromatography was conducted. After purifications, aproximately 5.6kD and 4.5kD proteins could be seen by Tricine-SDS-PAGE analysis. The two kinds of recombinant hPAB-βboth showed fine bacteriostasis activity towards S.aureus and P.aeruginosa clinical isolates in the assay.Fourthly, the conditions of high-density fermentation for hPAB-β. Compared with expression in flask, the growing conditions in high-density fermentation were quite different in culture medium, cell density, dissolved oxygen, etc. So, after expression in flask, the conditions in high-density fermentation for hPAB-βwithout tag were further approached: we conducted fed-batch high-density fermentation. Inoculated engineering bacteria in BMGY medium as the first grade bateria, and then inoculated it in 200ml BMGY as the second grade bacteria. Subsequently, inoculated the latter bacteria in 2L basal salts medium into the fermentation, adding trace salts medium into it. Controlled pO2 above 70%, pH among 5.0±0.05, tempretuer at 30℃, initial speed at 600r/min. When glycerine was exhausted, and pO2 went up, add growth medium to the fermenter at 10 ml/L per hour, until the wet weight reached 200～250g/L. After 3 hours not adding growth medium into the fermenter, add induction medium into it at 3 ml/L per hour for 3 hours. Then increased the speed to 9 ml/L per hour. According to these conditions, a 4.5 kD protein was expressed in the supernatant by Tricine-SDS-PAGE analysis. The total protein concentration in 3 fermentations were 664.0μg/ml, 781.8μg/ml and 721.3μg/ml. The expression level were 11.1%, 9.8%and 10.3%. Thus, the expression quantity were 73.7mg/L, 76.6 mg/L and 74.3mg/L.Finally, the purification of recombinant hPAB-βwithout tag after high-density fermentation and biological activity identification. The fermentation supernatant was first purified by reverse phase chromatography. After that, P10 gel filtration chromatography was conducted. Tricine-SDS-PAGE analysis showed that target peptide was in peak 2. Then, RP-HPLC was finally used, and the purity of taget peptide coule reach to 95%. The purified recombinant hPAB-βshowed bacteriostasis activity towards S.aureus clinical isolate(MRSA). And it’s antimicrobial activity was stronger than clindamycin and oxacillin, a little weaker than norvancomycin.Conclusion:We obtained natural and optimized sequence of hPAB-βwith 6×His tag and optimized sequence of hPAB-βwithout 6×His tag. Constructed two kinds of recombinant plasmids, screened multicopy transformants that coule express target peptides. The conditions of high-desity fermentation for hPAB-βwas groped. Then, recombinant hPAB-βwith antibacterial activity could be obtained after series of purifications.更多还原