本文選題：乳酸菌　切入點(diǎn)：分離鑒定　出處：《石河子大學(xué)》2013年碩士論文

【摘要】：乳鐵蛋白肽（Lactoferricin，Lfcin）是乳鐵蛋白（Lactoferrin，LF）經(jīng)胃蛋白酶水解，從N-端解離下來的一段氨基酸殘基，具有乳鐵蛋白除轉(zhuǎn)鐵功能之外幾乎全部的生物學(xué)活性，如廣譜抗菌、消炎、抑制腫瘤細(xì)胞生長及調(diào)節(jié)機(jī)體免疫反應(yīng)等。但是Lfcin在動物體內(nèi)含量極微，提取費(fèi)用昂貴，無法實(shí)現(xiàn)大規(guī)模生產(chǎn)，因此開展Lfcin基因工程研究具有重要意義。目前對乳鐵蛋白肽的研究多集中在原核表達(dá)上，但是由于LF及Lfcin對細(xì)菌的抗性作用，不能用原核表達(dá)系統(tǒng)直接表達(dá)具有生物學(xué)活性的LF或Lfcin，而乳鐵蛋白對于鐵需求低的乳酸菌沒有抑制作用，本研究自主設(shè)計(jì)并構(gòu)建大腸桿菌-乳酸菌雙復(fù)制子穿梭型豬乳鐵蛋白N-端亞基分泌表達(dá)載體，以具有自主知識產(chǎn)權(quán)的乳酸菌為呈遞系統(tǒng)，使目的蛋白得到成功表達(dá)。 從新疆、黑龍江等地采集各種凍土、野生禽類糞便等樣品材料，接種MRS培養(yǎng)基分離具有典型特征的乳酸菌，富集培養(yǎng)后提取乳酸菌的基因組DNA，利用細(xì)菌域通用引物擴(kuò)增16SrDNA序列，連接pMD-18T載體全部進(jìn)行測序，利用Geneious軟件進(jìn)行細(xì)菌的進(jìn)化樹分析，同時(shí)，采用法國梅里埃公司API50CHL鑒定試劑盒進(jìn)行碳水化合物發(fā)酵鑒定，篩選出的菌種，進(jìn)行接種SPF雛雞的增重試驗(yàn)，，每只雞隔天口服一次，口服菌量大約為6~8*108CFU，每周稱重，共6周。根據(jù)增重情況篩選出對雛雞有良好增重作用的乳酸菌2株（4151A1和411-15）；根據(jù)益生菌的篩選標(biāo)準(zhǔn)，對2株乳酸菌進(jìn)行耐酸、耐膽鹽、體外抗菌效果評價(jià)，最終篩選出4151A1命名為LactobacillussalvariusY1（L.salvariusY1）同時(shí)和實(shí)驗(yàn)室保存標(biāo)準(zhǔn)菌株L.casei393一起作為本研究的宿主菌。 利用本實(shí)驗(yàn)室已有條件，自主構(gòu)建乳糖誘導(dǎo)型表達(dá)豬乳鐵蛋白N-端亞基的大腸桿菌-乳酸菌雙復(fù)制子穿梭型分泌表達(dá)載體。首先進(jìn)行雙復(fù)制子穿梭載體的構(gòu)建，PCR擴(kuò)增質(zhì)粒pMB1的復(fù)制子，并與pHE1載體進(jìn)行克隆連接，得到pHE1-MB1雙復(fù)制子穿梭載體；然后以乳酸菌質(zhì)粒pPG612.1為模板擴(kuò)增其表達(dá)盒，與構(gòu)建的雙復(fù)制子載體克隆連接得到分泌表達(dá)載體pHE1-MB1-612；以pUC59-OPTF為模板擴(kuò)增出密碼子優(yōu)化的豬乳鐵蛋白N-端亞基（OPTF-N），克隆至pHE1-MB1-612上，得到第一個(gè)表達(dá)豬乳鐵蛋白N-端亞基的穿梭型雙復(fù)制子分泌表達(dá)載體pHE1-MB1-612-OPTF-N；再以pHE1-MB1-612-OPTF-N為模板，擴(kuò)增OPTF-N表達(dá)盒（3700bp），克隆至氯霉素抗性雙復(fù)制子穿梭型載體pGBHC-MB1上，得到第二個(gè)表達(dá)OPTF-N載體pHC-MB1-612-OPTF-N。將構(gòu)建完成的2個(gè)載體電擊轉(zhuǎn)化至自主分離的4151A1和標(biāo)準(zhǔn)菌株L.casei393，得到四株重組乳酸菌，分別命名為r-LactobacillussalvariusY1HC-OPTF-N、r-Lactobacilluscasei393HC-OPTF-N、r-LactobacillussalvariusY1HE1-OPTF-N、r-Lactobacilluscasei393HE1-OPTF-N。將4株重組菌和2株天然菌株接種至乳糖MRS進(jìn)行乳糖誘導(dǎo)表達(dá)，表達(dá)產(chǎn)物用免疫斑點(diǎn)實(shí)驗(yàn)進(jìn)行檢測，重組乳酸菌發(fā)生特異性顯色反應(yīng)，表明目的蛋白在4151A1和L.casei393中成功表達(dá)。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：S852.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 唐麗杰;哈卓;趙麗麗;宗曉淋;劉荻

本文編號：1663685