摘要
中国对虾是我国重要的海产养殖品种,具有重要的经济地位。大规模的养殖使对虾传染性疾病频发,造成重大经济损失,因而,找到有效预防和诊断治疗对虾疾病的方法迫在眉睫。利用一系列的生物化学和分子生物学方法,研究对虾先天免疫相关基因C-型凝集素及其功能,能够在一定层面上提供对虾免疫机制的认识,为对虾疾病的防治提供理论依据。针对海水养殖业病害发生的现状,从筛选和克隆海洋动物抗病功能基因——抗菌肽基因入手,应用基因工程手段获得抗菌肽融合基因,以期研制安全、高效、广谱的抗细菌、抗病毒的重组抗病蛋白渔药,从而减少养殖病害发生,提高水产品质量,保障水产养殖业可持续发展。
本论文主要报道了中国明对虾先天免疫中重要的模式识别蛋白(patternrecognition proteins,PRPs)C-型凝集素的重组表达及功能分析;利用基因工程手段获得融合表达抗菌肽(Antimicrobial peptides,AMPs)并研究其抗菌功能。主要结果如下:
1.中国明对虾C-型凝集素的重组表达和功能分析
无脊椎动物通过模式识别受体识别病原微生物细胞表面特异保守的糖分子,从而能够识别入侵微生物。C-型凝集素属于模式识别蛋白,在对虾先天免疫中起重要作用。本实验室从中国明对虾中克隆得到一种新型的C-型凝集素,命名为Fc-hsL,并发现其与免疫联系紧密。
本论文在原核表达系统中大规模诱导表达重组蛋白Fc-hsL,利用纯化的重组蛋白进行了一系列性质研究。凝集实验证明,Fc-hsL成熟肽在钙离子存在情况下可以凝集革兰氏阳性菌和革兰氏阴性菌,包括:巨大芽孢杆菌,大肠杆菌等。细菌结合实验发现,重组蛋白可以结合巨大芽孢杆菌,苏云金芽孢杆菌,藤黄微球菌和金黄色葡萄球菌等革兰氏阳性菌,以及部分革兰氏阴性菌,如革兰氏阴性菌大肠杆菌和肺炎克雷伯氏杆菌。通过进一步实验发现,Fc-hsL对甘露糖、半乳糖、脂多糖等都有结合能力,对甘露糖的结合能力大于对半乳糖的结合能力。通过管碟法和液体生长抑制实验等方法测试重组蛋白的抑菌活性和最小抑菌浓度(MIC),发现其对革兰氏阳性菌和真菌的抑菌能力较强,对革兰氏阴性菌有一定的抑菌能力。
2.融合抗菌肽表达载体构建、重组表达和性质分析
抗菌肽(Antimicrobial peptides,AMPs)是先天免疫系统重要的组成成分。本实验室从家蝇(Musca domestica)中克隆得到的防御素(Musca domesticadefensin,Mdde)具有抗革兰氏阳性菌和部分革兰氏阴性菌的活性(Wang et al.,2006)。中国明对虾对虾素(penaeidin 5)是本实验室从其血细胞中克隆得到的,对革兰氏阳性菌,阴性菌和真菌有抗性,最小抑菌浓度(MIC)分别:6.3-25.0μM,12.5-50μM,6.25-12.5μM(Kang et al.,2007)。
本论文通过重叠延伸PCR的方法将中国明对虾对虾素-5(penaeidin 5)和家蝇防御素Mdde基因片段融合,构建表达载体pET30a-pen-mdde。这个方法的核心就在于设计两对引物。首先位于融合基因后端的Mdde的前引物引入penaeidin5末端的22个核苷酸;其次penaeidin 5的前引物和Mdde的后引物引入EcoR1和XhoI酶切位点。本论文成功地在大肠杆菌中大规模诱导表达,并纯化得到了融合蛋白。利用管碟法和液体生长抑制法进行检测,发现重组蛋白有抗菌活性,对革兰氏阳性菌,阴性菌和真菌的最小抑菌浓度分别为:0.3-2.4μM,1.2-2.4μM,0.3-2.7μM。和单独的抗菌肽相比,融合蛋白对革兰氏阳性菌和革兰氏阴性菌以及真菌的抗性都大大增强,例如:对虾素-5抑制革兰氏阳性菌,阴性菌和真菌所需最小的浓度分别为:6.3μM,12.5μM,6.25μM(Kang et al.,2007),而融合蛋白重组蛋白为:0.3μM,1.2μM,0.3μM,其中,对真菌苹果炭疽菌(Glomerella album)和革兰氏阳性菌苏云金杆菌(Bacillus thuringiensis)的最小抑菌浓度为0.3μM。而且融合蛋白保留了对虾素penaeidin 5和家蝇防御素Mdde对革兰氏阳性菌的抗性大于对革兰氏阴性菌的抗性的特点。
As one of important mariculture varieties,Fenneropenaeus chinensis has an important economic status.Large-scale shrimp farming have caused frequent infectious diseases,which resulted in heavy economic losses.So it is high time that we find a effective way to prevent,diagnose and treat shrimp disease.The research on innate immune-related genes and their function of Chinese shrimp,using of a series of biochemistry and molecular biology methods,provides knowledges about shrimp immune mechanism and theoretical basis for disease control.Encountering the situation of mariculture industry,we take screening and(?)loning marine animals' disease-resistant gene(Antimicrobial peptides,AMPs)as a start,acquiring a fusion gene of AMPs by means of genetic engineering technology.The aim is to develop a kind of fishery drugs which is safe,efficient and broad-spectrum anti-bacterial and anti-viral activities.At the same time,we expect to reduce the frequency of mariculture disease and to improve the quality of aquatic products.
My studies focus on the recombinant protein expression and functions analysis of C-type lectin,which belongs to the PRPs family.The second part is about getting a fusion AMP protein of the defensin from housefly and the penaeidin 5 from shrimp and testing its antimicrobial activity.
1.Recombinant expression and functions analysis of a C-type lectin
Invertebrates recognize non-self with pattern recognition receptors,which recognize conserved and specific molecules on the surface of invading microorganisms.C-type lectin which palys an important role in the shrimp innate immune,is a member of PRPs family.One novel C-type lectin named Fc-hsL has been cloned from Chinese shrimp.
In our studies,we got high expression and purification of recombinant protein of Fc-hsL in E.coli.The recombinant Fc-hsL had agglutinating,binding and antimicrobial activities in vitro.Recombinant mature Fc-hsL had agglutinating activity against Gram-positive and-negative bacteria,including B.megaterium,E. coli.with calcium.Recombinant Fc-hsL bound to several Gram-positive bacteria, such as B.cereus,B.megaterium,B.thuringiensis,M.luteus and S.aureus,and some Gram-negative bacteria,such as Escherichia coli,Klebsiella pneumoniae.Various sugars can be bound by recombinant Fc-hsL.For example,mannose,glucose,LPS can inhibit the agglutination of recombinant Fc-hsL to B.megaterium cells.The antimicrobial activity of Fc-hsL recombinant protein was tested by Oxford cup method and minimal inhibitory concentration(MIC)assay,the results showed high antimicrobial activity against some Gram-positive bacteria and fungi,however displayed comparatively low antimicrobial activity of Gram-negative bacteria.
2.Expression vector construction,recombinant expression and functions analysis of a fusion antimicrobial peptide.
The AMPs is extremely important in the innate immunity.Previously,we have got a defensin from Musca domestica named Musca domestica defensin(Mdde), which has activity against Gram-positive and-negative bacteria(Wang et al.,2006) and penaeidin 5 from Chinese shrimp(Kang et al.,2007).
Two pairs of primers was designed for the overlap extension PCR.The last 22 nucleotides of penaeidin 5 were introduced by the former primer of Mdde which was at the back-end of the fusion gene.T wo restriction sites(EcoRI and X hoI)were introduced by the fomer forward primer of penaeidin 5 which was in front of the fusion gene and the reverse primer of Mdde.The fusion gene was amplified and recombinant expression vector pET30a-pen-mdde was constructed.The recombinant vector pET30a-pen-mdde was transfored into E.coli.The recombinant fusion AMP, Pen-Mdde,was successfully expressed and purified.It was found that the recombinant protein had antimicrobial activity against Gram-positive,Gram -negative bacteria and fungi by Oxford cup method and minimal inhibitory concentration assay.The MIC of rPen-Mdde against Gram-positive,Gram-negative bacteria and fungi was 0.3-2.4μM,1.2-2.4μM,0.3-2.7μM respectively.It was also found that the antimicrobial activity of the fusion gene recombinant protein had been greatly enhanced compared with the the recombinant protein of penaeidin 5 and Mdde.For example,the MIC of the penaeidin 5 which can inhibit the growth of Gram-positive,Gram-negative bacteria and fungi was 6.3μM,12.5μM,6.25μM(Kang et al.,2007).However,the MIC of fusion gene recombinant protein was 0.3μM,1.2μM,0.3μM.It was especially that the MIC against Glomerella album and Bacillus thuringiensis was all 0.3μM.And the characteristics of having a better resistance activity against Gram-positive than Gram-negative which was also had by penaeidin 5 and Mdde was maintained.
引文
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