益生性乳酸菌的分离鉴定及表达猪乳铁蛋白基因重组乳酸菌的构建

发布时间：2018-03-25 15:16

本文选题：乳酸菌　切入点：分离鉴定　出处：《石河子大学》2013年硕士论文

【摘要】：乳铁蛋白肽（Lactoferricin，Lfcin）是乳铁蛋白（Lactoferrin，LF）经胃蛋白酶水解，从N-端解离下来的一段氨基酸残基，具有乳铁蛋白除转铁功能之外几乎全部的生物学活性，如广谱抗菌、消炎、抑制肿瘤细胞生长及调节机体免疫反应等。但是Lfcin在动物体内含量极微，提取费用昂贵，无法实现大规模生产，因此开展Lfcin基因工程研究具有重要意义。目前对乳铁蛋白肽的研究多集中在原核表达上，但是由于LF及Lfcin对细菌的抗性作用，不能用原核表达系统直接表达具有生物学活性的LF或Lfcin，而乳铁蛋白对于铁需求低的乳酸菌没有抑制作用，本研究自主设计并构建大肠杆菌-乳酸菌双复制子穿梭型猪乳铁蛋白N-端亚基分泌表达载体，以具有自主知识产权的乳酸菌为呈递系统，使目的蛋白得到成功表达。从新疆、黑龙江等地采集各种冻土、野生禽类粪便等样品材料，接种MRS培养基分离具有典型特征的乳酸菌，富集培养后提取乳酸菌的基因组DNA，利用细菌域通用引物扩增16SrDNA序列，连接pMD-18T载体全部进行测序，利用Geneious软件进行细菌的进化树分析，同时，采用法国梅里埃公司API50CHL鉴定试剂盒进行碳水化合物发酵鉴定，筛选出的菌种，进行接种SPF雏鸡的增重试验，，每只鸡隔天口服一次，口服菌量大约为6~8*108CFU，每周称重，共6周。根据增重情况筛选出对雏鸡有良好增重作用的乳酸菌2株（4151A1和411-15）；根据益生菌的筛选标准，对2株乳酸菌进行耐酸、耐胆盐、体外抗菌效果评价，最终筛选出4151A1命名为LactobacillussalvariusY1（L.salvariusY1）同时和实验室保存标准菌株L.casei393一起作为本研究的宿主菌。利用本实验室已有条件，自主构建乳糖诱导型表达猪乳铁蛋白N-端亚基的大肠杆菌-乳酸菌双复制子穿梭型分泌表达载体。首先进行双复制子穿梭载体的构建，PCR扩增质粒pMB1的复制子，并与pHE1载体进行克隆连接，得到pHE1-MB1双复制子穿梭载体；然后以乳酸菌质粒pPG612.1为模板扩增其表达盒，与构建的双复制子载体克隆连接得到分泌表达载体pHE1-MB1-612；以pUC59-OPTF为模板扩增出密码子优化的猪乳铁蛋白N-端亚基（OPTF-N），克隆至pHE1-MB1-612上，得到第一个表达猪乳铁蛋白N-端亚基的穿梭型双复制子分泌表达载体pHE1-MB1-612-OPTF-N；再以pHE1-MB1-612-OPTF-N为模板，扩增OPTF-N表达盒（3700bp），克隆至氯霉素抗性双复制子穿梭型载体pGBHC-MB1上，得到第二个表达OPTF-N载体pHC-MB1-612-OPTF-N。将构建完成的2个载体电击转化至自主分离的4151A1和标准菌株L.casei393，得到四株重组乳酸菌，分别命名为r-LactobacillussalvariusY1HC-OPTF-N、r-Lactobacilluscasei393HC-OPTF-N、r-LactobacillussalvariusY1HE1-OPTF-N、r-Lactobacilluscasei393HE1-OPTF-N。将4株重组菌和2株天然菌株接种至乳糖MRS进行乳糖诱导表达，表达产物用免疫斑点实验进行检测，重组乳酸菌发生特异性显色反应，表明目的蛋白在4151A1和L.casei393中成功表达。
[Abstract]:Lactoferrin peptide Lactoferrin (Lfcin) is a segment of amino acid residues which is hydrolyzed by pepsin and dissociated from the N-terminal of lactoferrin. It has almost all the biological activities of lactoferrin except transferrin, such as broad-spectrum antibacterial and anti-inflammatory. Inhibition of tumor cell growth and regulation of immune response, etc. However, Lfcin content in animals is very small, and the cost of extraction is too high to achieve large-scale production. Therefore, it is of great significance to study Lfcin gene engineering. At present, most of the studies on lactoferrin peptides focus on prokaryotic expression, but because of the resistance of LF and Lfcin to bacteria, The prokaryotic expression system could not directly express LF or Lfcinin with biological activity, but lactoferrin had no inhibitory effect on lactic acid bacteria with low iron demand. In this study, we designed and constructed Escherichia coli-lactic acid bacteria double replicator shuttle porcine lactoferrin N-terminal subunit secretory expression vector, using lactic acid bacteria with independent intellectual property as the presentation system, so that the target protein was successfully expressed. Samples of permafrost and wild poultry faeces were collected from Xinjiang, Heilongjiang and other places, and lactic acid bacteria with typical characteristics were isolated from MRS medium. The genomic DNA of lactic acid bacteria was extracted after enrichment and culture. The 16SrDNA sequence was amplified by universal primers in bacterial domain, and the pMD-18T vector was ligated for sequencing. The phylogenetic tree of the bacteria was analyzed by Geneious software. At the same time, Carbohydrate fermentation was carried out with the API50CHL identification kit of Merier, France. The selected strains were tested for weight gain by inoculating SPF chicks. Each chicken was given orally once every other day, and the amount of bacteria taken orally was about 6m 8108CFU, weighing every week. For 6 weeks, 2 strains of lactic acid bacteria with good weight gain were screened out according to the weight gain conditions, and 2 strains of lactic acid bacteria were selected according to the screening criteria of probiotics, and the in vitro antibacterial effect of 2 strains of lactic acid bacteria were evaluated according to the screening criteria of probiotics. Finally, 4151A1 was named Lactobacillus salvarius Y1 L.salvarius Y1) and the standard strain L.casei393 was used as the host of this study. Using the conditions already available in our laboratory, Lactose inducible double replicator shuttle vector expressing porcine lactoferrin N-terminal subunit was constructed. Firstly, the double replicon shuttle vector was constructed to amplify the replicon of the plasmid pMB1. The double replicon shuttle vector of pHE1-MB1 was obtained by cloning and ligating with pHE1 vector, and then the expression cassette was amplified by using lactic acid bacteria plasmid pPG612.1 as template. The secretory expression vector pHE1-MB1-612 was obtained by ligating with the constructed double replicon vector, and the codon optimized porcine lactoferrin N-terminal subunit (OPTF-NV) was amplified by using pUC59-OPTF as template, and cloned into pHE1-MB1-612. The first shuttle double replicon secreting vector pHE1-MB1-612-OPTF-Nwas obtained, which expressed porcine lactoferrin N-terminal subunit, and the OPTF-N expression cassette was amplified by using pHE1-MB1-612-OPTF-N as template, and cloned into chloramphenicol resistant double replicon shuttle vector pGBHC-MB1. The second OPTF-N vector pHC-MB1-612-OPTF-Nwas obtained. The two vectors were transformed into 4151A1 and standard strain L.casei393.The four recombinant lactic acid bacteria were obtained. They were named r-Lactobacillus salvariusY1HC-OPTF-Nr-Lactobacillus casei393HC-OPTF-Nr-Lactobacillus salvariusY1HE1-OPTF-N. four recombinant bacteria and two natural strains were inoculated into lactose MRS for lactose induced expression. The results showed that the target protein was successfully expressed in 4151A1 and L.casei393.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S852.4

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