【作者】 陈茜；

【导师】 高荣；

【作者基本信息】 四川大学 ， 遗传学， 2007， 硕士

【摘要】 溶菌酶（lysozyme，LYZ）是一种专门作用于微生物细胞壁的水解细菌细胞壁中粘多糖β-1，4-糖苷键的胞壁质酶，是一种碱性蛋白质，存在于许多动物、植物和微生物中。研究发现，溶菌酶对革兰氏阳性细菌具有直接的溶解作用，催化革兰氏阳性菌细胞壁不溶性多糖水解，生成可溶性黏肽。在分泌型免疫球蛋白A和补体的参与下，溶菌酶还对革兰氏阴性细菌具有间接的溶解作用，是一种天然的抗菌物质。由于其能够增强巨噬细胞和白细胞的吞噬和消化功能、将诱发炎症的酸性物质灭活、增强抗菌素及其它药物的疗效，具有消炎消肿、帮助组织修复、改善组织局部血液循环和分解脓液等药理作用，因此溶菌酶在医疗界中倍受关注。近年来，随着生物学技术和免疫学技术的发展，对溶菌酶的研究取得了令人瞩目的成就。利用DNA重组技术发展溶菌酶的抗菌效果，已成为其中重要的研究方向。牛体内溶菌酶（bovine lysozyme，bLys）有大约10种，表达的潜在位点有：乳腺、唾液、泪液、胃、气管组织以及中性粒细胞、嗜红细胞、巨噬细胞、白细胞等。由于牛奶与人们的生活息息相关，其质量的好坏关系着人们的健康，因此，对牛奶溶菌酶的研究尤为突出。为构建具有高效抗菌、活性持久的抗菌基因，探索研制新型抗菌制剂，克服耐药性细菌感染日益严重的问题，本实验构建了原核表达载体pGEX-4T-1-Lys（pGL）。本实验是以成功构建的新型牛溶菌酶基因原核表达载体pGL为模板，用PCR扩增得到目的片段Lys基因，将VR1020和目的片段Lys分别用BamHI和BglⅡ双酶切，并将VR1020酶切片段先进行去磷酸化，然后将两种酶切产物在16℃水浴过夜连接；连接产物转化大肠杆菌DH5α感受态细胞，筛选重组子，用碱裂解法小规模提取重组子质粒DNA，用BamHI，BglⅡ作双酶切鉴定验证，并作质粒PCR鉴定。经双酶切和质粒PCR的实验结果初步确定的阳性重组子进行双向基因测序，测序结果证明插入目的片段正确，证实新型牛溶菌酶基因已经克隆到真核表达载体VR1020（VR1020-Lys，命名为VRL）上。将重组质粒VRL以JetPEI^TM Cationic polymer分子包裹，介导转染真核HEK293细胞，转染培养24h、48h、72h后分别提取培养细胞mRNA进行RT-PCR，与VR1020转染细胞比较发现，转染后24h、48h和72h的HEK293细胞的RNA中含有与目的cDNA片段对应的mRNA。为了探索一种更经济高效的转染介质，本实验同时还制备了壳聚糖纳米颗粒（chitosan nanoparticles，CNP），用此壳聚糖纳米颗粒包裹重组质粒VRL后转染HEK293细胞，转染的HEK293细胞中也表达与目的cDNA片段对应的mRNA，说明了壳聚糖纳米颗粒也能作为一种有效的转染介质。用转染后HEK293细胞上清分别作抑制标准大肠杆菌、产毒性大肠杆菌、绿脓杆菌、金黄色葡萄球菌和肺炎球菌生长实验，实验结果证明新构建的真核表达质粒VRL具有良好的抑制革兰氏阳性菌生长的效果，这些为深入开展VRL在动物体内的抗感染抑菌实验奠定了可靠的基础。更多还原

【Abstract】 Lysozyme, which was recognized as bacteriolytic agent having an ability to hydrolyze bacterial cell walls, is found in a variety of vertebrate cells and secretions, such as spleen, milk, tears and egg white. It lyses certain bacteria by hydrolysing theβ-linkages between the muramic acid and N-acetylglucosamine of the mucopolysaccharides which are present in the bacterial cell wall. It has become one of the most intensively studied proteins. The action of Lysozyme on bacteria works cooperatively and synergistically with antibiotics, which has a very important practical value in medicine area. In present, the research of lysozyme develops greatly because of the development of the technology of biology and immunology. And it becomes very import that using the technology of DNA recombination to boost the antibacterial effect of lysozyme.There are about ten kinds of bovine lysozyme that are potentially produceded in neutrophil and macrophage in mammary gland, stomach and tracheal tissue, and secreted into saliva, tears fluid and milk which is an important food for human beings. In order to construct a novel lysozyme gene to improve its antibacterial activity and develop effective medicine to overcome the increasing infection of animals by antibiotic resistant bacteria, the experiment was carried out to reconstruct the eukaryotic expression vector for bovine milk lysozyme gene （Lys） by inserting theLys cDNA fragment into VR1020 plasmid. The VR1020 was digested by BamHⅠand BglⅡ, and then ligated with Lys cDNA digested wit hthe same double endoenzymes at 16℃overnight. The ligated products were used to transform E. coli DH5αcompetent cell, and the positive recombinant was screend out by inoculation of transformed cells in Lurea Bertani （LB） broth with kanamycin at 100μg/ml and shaked for 16 hours at 37℃. Bacterial cells were pelleted by centrifugation and plasmid DNA extracted following large-scale alkine lysis. Then the recombinant plasmid was digested by BamHⅠand BglⅡand plasmid PCR to identify the cloned Lys gene. Through DNA sequencing, it was proved that the Lys cDNA fragment was successfully inserted into the eukaryotic plasmid VR1020 in correct orientation.The ability of VRL to express Lys in vitro was assayed by RT-PCR in eukaryotic cells HEK293 that were transiently transfected with VRL wraped in JetPEI^TM Cationic polymer transfection reagent and chitosan nanoparticles （CNP）. The result was found that the mRNA was detected in the the total RNA of HK293 cells transfected after 24, 48, 72h; It was also showed that the expression effect of the gene enwrapped by CNP was the same with or better than that of JetPEI^TM Cationic polymer, indicating CNP is practicable package to facilitate the plasmid transfectection of eukaryotic cells. The expression products in the supernatant of transfected cell medium manifested significant inhibitary effect to suppress the growth of Streptococcus pneumoniae and Staphylococcus aureus bacteria in vitro（P＜0.05）, and this suggest that the recombinant eukaryotic plasmids of novel bovine lysozyme gene could be further developed into effective antibacterial reagent to control animal infection.更多还原