【作者】 王静；

【导师】 高利； 冯永谦；

【作者基本信息】 东北农业大学 ， 兽医博士（专业学位）， 2019， 博士

【摘要】 奶牛乳房炎是奶牛养殖业中最常见的、最高发的和能引起严重损失的奶牛疾病之一。奶牛乳房炎不仅能降低奶牛的奶产量和牛奶的品质,还能引起严重的食品安全问题。早期对乳房炎的防治,主要是使用抗生素。但是,因为滥用和长期使用抗生素,在治疗奶牛乳房炎的同时,也出现了一些其他的重要问题,如细菌产生了对抗生素的耐药性,影响治疗效果和牛奶中有过多的抗生素的残留而引起的食品安全问题等。目前,在奶牛乳房炎的治疗上,非抗生素物质的使用越来越多,而非抗生素物质也逐渐已成为治疗奶牛乳房炎的主要药物,其治疗策略也逐步替代以前的抗生素治疗。抗菌肽(Antibacterial peptide,ABP),是生物体内普遍存在的一类具有强烈抗菌作用的小分子多肽,在机体抵抗病原入侵方面起着重要作用。抗菌肽因其独特的抗菌机制和广谱、高效的抗菌活性而引起普遍关注。目前,运用基因工程方法表达抗菌肽基因,用于畜牧业中以防治家禽和家畜的某些疾病是抗菌肽应用的热点,也是治疗奶牛乳房炎的安全有效的手段之一。本研究通过重叠延伸PCR克隆了重组牛LAP-CATHL2抗菌肽,构建了它的原核、酵母和奶牛乳腺细胞3种表达载体,表达并纯化了重组牛LAP-CATHL2抗菌肽,并检测了重组牛LAP-CATHL2抗菌肽的抗菌谱及最小抑菌浓度,分析了它的序列结构特性。此外,本研究还通过构建奶牛乳房炎动物模型,检测了重组牛LAP-CATHL2抗菌肽对奶牛乳房炎的防治效果。本研究首先从牛舌鳞状上皮细胞和股骨骨髓细胞中分别克隆出牛β-防御素LAP和牛CATHL2抗菌肽,然后利用重叠延伸PCR克隆得到重组牛LAP-CATHL2抗菌肽。本研究将重组牛LAP-CATHL2抗菌肽分别克隆进pGEX-4T-1载体、pPICZαA载体、和插入了αS1酪蛋白调控序列作为启动子的pCMV-C-Flag载体,分别构建了重组牛LAP-CATHL2抗菌肽的原核表达载体、酵母表达载体和奶牛乳腺细胞表达载体,并将这些表达载体分别转进感受态大肠杆菌BL21、毕赤酵母GS115和分离纯化的原代奶牛乳腺细胞中。对这些表达系统中的重组牛LAP-CATHL2抗菌肽进行诱导表达,并纯化得到了重组牛LAP-CATHL2抗菌肽。其产量分别是2.83、1.16、0.114 mg/mL培养液。用包括革兰氏阴性菌、革兰氏阳性菌和真菌在内的金黄色葡萄球菌(Staphylococcus aureus ATCC25923)、致病性大肠杆菌(Escherichia coli ATCC25922)、无乳链球菌(S.agalactiae)、乳房链球菌(Streptococcus uberis)、停乳链球菌(Streptococcus dysgalactiae)、单核细胞增生李斯特氏菌(Listeria monocytogenes NICPBP54002)、克雷伯氏菌(Klebsiella spp.)、蜡样芽孢杆菌(Bacillus cereus ATCC10987)和白色念珠菌(Nostoc)等9种菌对重组牛LAP-CATHL2抗菌肽的抗菌谱进行分析。结果表明,重组牛LAP-CATHL2抗菌肽具有广泛的抗菌谱,对革兰氏阴性菌、革兰氏阳性菌和真菌均有明显的抑菌效果,在9种测试菌中尤其对金黄色葡萄球菌、致病性大肠杆菌、无乳链球菌和白色念珠菌的抑菌能力最强。用金黄色葡萄球菌、致病性大肠杆菌、无乳链球菌和白色念珠菌检测了3个不同表达系统表达的重组牛LAP-CATHL2抗菌肽的最低抑菌浓度。结果表明,原核表达系统表达的此抗菌肽活性最低,其对4种测试菌的最小抑菌浓度依次是500μg/mL、250μg/mL、250μg/mL和500μg/mL;酵母表达载体表达的抗菌肽对4种测试菌的最低抑菌浓度分别是125μg/mL、62.5μg/mL、62.5μg/mL和125μg/mL;奶牛乳腺上皮细胞表达载体表达的抗菌肽对4种测试菌的最低抑菌浓度分别是31.25μg/mL、31.25μg/mL、31.25μg/mL和62.5μg/mL。用生物信息学软件和网站预测分析了重组牛LAP-CATHL2抗菌肽的亲/疏水性、跨膜区域、信号肽结构、磷酸化位点、氨基酸序列一级结构、二级结构和三级结构等结构信息。结果表明,重组牛LAP-CATHL2抗菌肽既有亲水性部分也有疏水性部分,属于两亲性蛋白。此外,其序列中有明显的跨膜区域和多个可能的磷酸化位点;在二级和三级结构中,此抗菌肽的二级结构主要包含β-折叠,有多个正向或反向的β-折叠区域,各个β-折叠区域以其两端的松散的成环状的肽链连接。利用致病性大肠杆菌和金黄色葡萄球菌感染,构建了奶牛乳房炎的动物模型。通过乳房灌注试验和乳房注射质粒试验检测了重组牛LAP-CATHL2抗菌肽对奶牛乳房炎的防治效果。结果表明,乳房灌注时,当抗菌肽浓度为250 mg/mL,每个乳区每天灌注10 mL抗菌肽溶液时,酵母和奶牛乳腺细胞表达系统表达的重组牛LAP-CATHL2抗菌肽对奶牛乳房炎均有很好的治疗效果,而原核系统表达的抗菌肽治疗效果不明显;乳房注射质粒时,注射奶牛乳腺细胞表达系统的质粒对奶牛乳房炎有很好的预防效果,而注射原核和酵母表达系统的质粒时,其预防效果不明显。本试验成功克隆了重组牛LAP-CATHL2抗菌肽,并构建了其原核、酵母和奶牛乳腺细胞3种表达质粒。在3种表达系统中均成功诱导表达了具有广谱抗菌活性的重组牛LAP-CATHL2抗菌肽,原核表达系统中表达的重组抗菌肽活性最低。此外,奶牛临床试验表明,酵母和奶牛乳腺细胞两种系统体外表达的重组抗菌肽对大肠杆菌和金黄色葡萄球菌的奶牛乳房炎有显著治疗效果,而在乳腺注射质粒时,只有奶牛乳腺细胞表达载体对奶牛乳房炎有显著防治效果。更多还原

【Abstract】 Cow mastitis is one of the most common and highest diseases in dairy industry,which can cause serious loss.Cow mastitis can not only reduce milk yield and milk quality,but also cause serious food safety problems.In the early stage,the prevention and treatment of mastitis was mainly the use of antibiotics peptide.However,due to the abuse and long-term use of antibiotics peptide,there are also other important problems in the treatment of dairy cow mastitis,such as bacterial resistance to antibiotics peptide,Effects on treatment and food safety caused by excessive antibiotic residues in milk.At present,in the treatment of dairy cow mastitis,the use of non-antibiotic substances is increasing,and non-antibiotic substances have gradually become the main drug for the treatment of dairy cow mastitis,and its treatment strategy has gradually replaced the previous antibiotic treatment.Antibiotics peptide(ABP)is a type of small molecule polypeptide that has a strong antibacterial effect and plays an important role in the body’s resistance to pathogen invasion.Antibiotics peptide have attracted widespread attention because of their unique antibacterial mechanism and wide spectrum and high efficiency antibacterial activity.At present,using genetic engineering method to express antimicrobial peptide gene to prevent and treat some diseases of poultry and livestock in animal husbandry is a hot spot in the application of antimicrobial peptide,and it is also one of the safe and effective methods to treat cow mastitis.In this study,the recombinant bovine LAP-CATHL2 antibiotic was cloned by overlapping extended PCR,and its prokaryotic,yeast and dairy cow mammary gland cell expression vectors were constructed.The recombinant cow LAP-CATHL2 antibiotic was expressed and purified,and the antibacterial spectrum and minimum antibacterial concentration of it were detected,and the sequence structure characteristics of it was analyzed.In addition,the effects of recombinant bovine LAP-CATHL2 antibiotics peptide on bovine mastitis were tested by constructing an animal model of bacterial cow mastitis.In this study,bovine beta-defensin LAP and bovine CATHL2 antibiotics peptide were cloned from bovine squamous epithelial cells and femoral bone marrow cells,respectively.And then,recombinant bovine LAP-CATHL2 antibiotics peptide were obtained by overlapping extended PCR cloning.In this study,recombinant cattle LAP-CATHL2 antibiotics peptide were cloned into p GEX-4T-1 vector,p PICZ αA vector,and p CMV-C-Flag vector contains an inserted regulatory sequence of αS1 casein as a promoter,respectively.And the prokaryotic expression vector,yeast expression vector and dairy cow mammary gland cell expression vector of recombinant bovine LAP-CATHL2 antibiotics peptide were constructed.These expression vectors were transferred to the Escherichia coli BL21,Pyretic yeast GS115 and purified primary cow mammary gland cells,respectively.The expression of recombinant bovine LAP-CATHL2 antibiotics peptide in these expression systems were induced and purified.The yield of recombinant bovine LAP-CATHL2 antibiotics peptide in the prokaryotic expression vector,yeast expression vector and dairy cow mammary gland cell expression vector is 2.83,1.16,and 0.114 mg/m L cultures,respectively.The antimicrobial spectra of recombinant bovine LAP-CATHL2 antibiotics peptide was analyzed by nine kinds of bacteria(Staphylococcus aureus ATCC25923,Escherichia coli ATCC25922,three kinds of Streptococcus(Sc.uberis,S.agalactiae,S.dysgalactiae equisimilis),Listeria monocytogenes NICPBP54002,Klebsiella spp,Bacillus cereus ATCC10987 and Nostoc)including gram-negative bacteria,gram-positive bacteria and fungi.The results showed that the recombinant bovine LAP-CATHL2 antibiotics peptide had a wide antimicrobial spectrum and had obvious antibacterial effect on gram-negative bacteria,gram-positive bacteria and fungi.Of the nine tested bacteria,the antibiotics peptide had the strongest antimicrobial resistance to Staphylococcus aureus,Escherichia coli,S.agalactiae and Nostoc.The Staphylococcus aureus,Escherichia coli,S.agalactiae and Nostoc were used to determine the minimum antibacterial concentration of recombinant bovine LAP-CATHL2 antibiotics peptide expressed by three different expression systems.The results showed that the antimicrobial activity of recombinant bovine LAP-CATHL2 antibiotics peptide expressed by prokaryotic expression system was the lowest,and the minimum antibacterial concentration for the four test bacteria was 500 μg/m L,250 μg/m L,250 μg/m L,and 500 μg/m L,respectively.The minimum antibacterial concentration of the recombinant bovine LAP-CATHL2 antibiotics peptide expressed by yeast expression system for four test bacteria was 125 μg/m L,62.5 μg/m L,62.5 μg/m L and 125 μg/m L,respectively.And the minimum antibacterial concentration of the recombinant bovine LAP-CATHL2 antibiotics peptide expressed by dairy cow mammary gland cell expression system for four test bacteria was 31.25 μg/m L,31.25 μg/m L,31.25 μg/m L and 62.5 μg/m L,respectively.The structure information of recombinant bovine LAP-CATHL2 antibiotics peptide such as hydrophilicity,transmembrane region,signal peptide structure,phosphorylation site,amino acid sequence primary structure,secondary structure and tertiary structure were analyzed using bioinformatics software and website prediction.The results show that the recombinant bovine LAP-CATHL2 antibiotics peptide have both hydrophilic and hydrophobic parts and belong to amphiphilic proteins.In addition,there are obvious transmembrane regions and multiple possible phosphorylation sites in the sequence.In the secondary and tertiary structures,the secondary structure of this antibiotic mainly consists of β-folding.It has multiple positive or reverse β-folding regions,and each β-folding region is connected by a loosely wound peptide chain at both ends.Using Staphylococcus aureus and Escherichia coli infection,an animal model of bacterial cow mastitis was constructed.The effects of recombinant bovine LAP-CATHL2 antibiotics peptide on bacterial cow mastitis were tested by breast perfusion and breast injection plasmid.The results showed that in the breast perfusion test,when the antibiotic concentration was 250 mg/m L and 10 m L antibiotic solution was perfusied daily in each breast area,the recombinant bovine LAP-CATHL2 antibiotics peptide expressed by yeast and dairy cow mammary gland cell expression system have a good therapeutic effect on bacterial cow mastitis,but the therapeutic effect of prokaryotic system expression was not obvious.When in the breast injection plasmid test,the injected plasmid from the dairy cow mammary gland cell expression system has a good preventive effect on bacterial cow mastitis but the injected plasmid from the prokaryotic and yeast expression system has no significant preventive effect.In summary,this study successfully cloned the recombinant bovine LAP-CATHL2 antibiotic,and constructed its prokaryotic,yeast and cow mammary gland cells to express plasmid.The recombinant bovine LAP-CATHL2 antibiotic was successfully induced and expressed in all three expression systems,and they all had broad spectrum antibacterial activity,but the antibacterial activity of recombinant antibiotics peptide expressed in prokaryotic expression system is the lowest.In addition,animal experiments have shown that recombinant antibiotics peptide expressed in vitro by yeast and dairy cow mammary cells have a significant effect on cow mastiti s caused by Staphylococcus aureus and Escherichia coli,but in the breast injection plasmid test,only the dairy cow mammary gland cell expression vector has a significant effect on the control of bacterial cow mastitis.更多还原