本文選題：雞β-防御素 + 乳酸菌��； 參考：《吉林農(nóng)業(yè)大學》2015年碩士論文

【摘要】：防御素抗菌譜廣,對人和動物無毒副作用,具有免疫原性低、無耐藥性、無殘留、溶解性和熱穩(wěn)定性好,對高離子強度和pH變化不敏感等特點。因此,防御素具有替代傳統(tǒng)抗生素的潛力。β-防御素是動物體內(nèi)一種重要的抗菌肽,不僅有抗病毒和殺菌的作用,而且它還參與機體的免疫調(diào)節(jié),有較高的研發(fā)利用潛力,是當前生物醫(yī)學的研究熱點之一。本研究以雞β-防御素6(Gal-6)為研究對象,根據(jù)Genebank中登錄的Gal-6序列合成Gal-6,構(gòu)建含有Gal-6的質(zhì)粒pUC57-T-Gal-6。將它和穿梭表達載體pSIP409同時進行酶切,連接構(gòu)建pSIP409-Gal-6質(zhì)粒,轉(zhuǎn)入大腸桿菌DH5α中進行鑒定,進而將鑒定正確的質(zhì)粒利用電轉(zhuǎn)化技術(shù)轉(zhuǎn)入乳桿菌感受態(tài)NC8中。制備重組Gal-6基因乳酸菌。通過SDS-PAGE、Western blotting方法檢測Gal-6在植物乳桿菌中的表達情況,應(yīng)用薄層平皿瓊脂糖擴散法測定重組乳酸菌的抑菌活性。結(jié)果顯示成功構(gòu)建了重組質(zhì)粒pSIP409-Gal-6,并利用電轉(zhuǎn)化技術(shù)成功轉(zhuǎn)入植物乳酸桿菌中,通過檢測驗證表達成功。抑菌活性實驗證明Gal-6分別對金黃色葡萄球菌和大腸桿菌具有一定的抑菌活性。為進一步分析重組乳酸菌的免疫保護性效果,驗證Gal-6是否能增強雞的機體免疫功能,增強抗病和抗感染能力,將構(gòu)建的重組乳酸菌pSIP409-Gal-6經(jīng)SppIP誘導表達后飼喂雛雞,45只2-3周齡的雛雞隨機分成3組,每組各15只,三組分別為pSIP409-Gal-6組,pSIP409組和PBS組,以重組乳酸菌5.0×109 CFU分組口服免疫雛雞,每只免疫計量控制為0.3ml,按以上計量進行免疫三周,第四周停止免疫,第五周再次進行免疫,免疫周期結(jié)束后對雞翅下采血并用流式細胞術(shù)檢測CD4+和CD8+T淋巴細胞。口服免疫結(jié)束2周后用致病性大腸桿菌ETEC 5.0×1010 CFU進行攻毒實驗。攻菌后每天監(jiān)測體重,檢測各組實驗雞的體重變化。攻毒后第10天處死,取十二指腸、回腸、盲腸制作石蠟包埋組織切片,H-E染色后觀察腸道病理變化。結(jié)果顯示,pSIP409-Gal-6組和pSIP409組的體重丟失低于PBS組。從流式細胞儀檢測結(jié)果看出,Gal-6能夠增加CD4+T淋巴細胞數(shù)量,增強機體免疫力。攻毒后腸道病理切片得出,口服重組乳酸菌pSIP409-Gal-6能改善腸道病理變化。因此,將β-防御素通過乳酸菌在動物體內(nèi)表達,增強動物機體抗感染能力,這對動物性食品安全和公共衛(wèi)生具重要意義,對減少和替代抗生素的使用起到積極促進作用。
[Abstract]:Defensin has a wide spectrum of antimicrobial spectrum and has no side effects on human and animal. It has the characteristics of low immunogenicity, no drug resistance, no residue, good solubility and thermal stability, and is not sensitive to the changes of high ionic strength and pH. Therefore, defensin has the potential to replace traditional antibiotics. And it is also involved in the immune regulation of the body, and it is also involved in the immune regulation of the body. It is one of the hot spots in biomedicine. This study takes chicken beta defensin 6 (Gal-6) as the research object, and syntheses Gal-6 according to the Gal-6 sequence of the login in Genebank, and constructs the plasmid pUC57-T-Gal-6. containing Gal-6 to express it and shuttle expression. At the same time, the body pSIP409 was cut through the enzyme, connected with the construction of the pSIP409-Gal-6 plasmid, and transferred into the Escherichia coli DH5 alpha. Then the identification of the correct plasmid was transferred into the Lactobacillus receptive NC8 by electrical transformation technology. The recombinant Gal-6 gene was prepared. The expression of Gal-6 in Lactobacillus plantarum was detected by SDS-PAGE and Western blotting method. The bacteriostasis activity of recombinant lactic acid bacteria was determined by the thin plate agarose diffusion method. The results showed that the recombinant plasmid pSIP409-Gal-6 was successfully constructed and successfully transferred into the Lactobacillus plantarum by electric conversion technology and proved to be successful through detection. The bacteriostasis experiment showed that Gal-6 was divided into Staphylococcus aureus and Escherichia coli. In order to further analyze the protective effect of the recombinant lactic acid bacteria, it is verified whether Gal-6 can enhance the immune function of the chicken body, enhance the disease resistance and anti infection ability. The recombinant Lactobacillus pSIP409-Gal-6 is induced by SppIP to feed the chicks, and 45 chicks at 2-3 weeks of age are randomly divided into 3 groups, 15 in each group and three in each group. In group pSIP409-Gal-6, group pSIP409 and group PBS, the immune chicks were immunized with recombinant Lactobacillus 5 x 109 CFU group, each immunometric control was 0.3ml, immunized for three weeks by the above measurement, the immunization in the fourth week was stopped, and the immunization was repeated for fifth weeks. After the immunization period, the blood was collected under the chicken wings and the CD4+ and CD8+T lymph were detected by flow cytometry. Cells. After 2 weeks of oral immunization, the attack was carried out with the pathogenic Escherichia coli ETEC 5 x 1010 CFU. After the attack, the body weight was monitored and the body weight changes were detected in each group. After tenth days of attack, the paraffin embedded tissue sections were made from the duodenum, the ileum and the cecum. The pathological changes of the intestines were observed after H-E staining. The results showed that pSIP409 The loss of body weight in group -Gal-6 and group pSIP409 was lower than that in group PBS. The results of flow cytometry showed that Gal-6 could increase the number of CD4+T lymphocytes and enhance the immunity of the body. After the attack of toxin, the oral recombinant lactic acid bacteria pSIP409-Gal-6 could improve the pathological changes of the intestine. It is of great significance to animal food safety and public health, and plays an active role in reducing and replacing antibiotics.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S859.79

【共引文獻】

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