鸡β防御素重组乳酸菌的制备及免疫保护效果研究

发布时间：2018-07-01 17:18

本文选题：鸡β-防御素 + 乳酸菌　；参考：《吉林农业大学》2015年硕士论文

【摘要】：防御素抗菌谱广,对人和动物无毒副作用,具有免疫原性低、无耐药性、无残留、溶解性和热稳定性好,对高离子强度和pH变化不敏感等特点。因此,防御素具有替代传统抗生素的潜力。β-防御素是动物体内一种重要的抗菌肽,不仅有抗病毒和杀菌的作用,而且它还参与机体的免疫调节,有较高的研发利用潜力,是当前生物医学的研究热点之一。本研究以鸡β-防御素6(Gal-6)为研究对象,根据Genebank中登录的Gal-6序列合成Gal-6,构建含有Gal-6的质粒pUC57-T-Gal-6。将它和穿梭表达载体pSIP409同时进行酶切,连接构建pSIP409-Gal-6质粒,转入大肠杆菌DH5α中进行鉴定,进而将鉴定正确的质粒利用电转化技术转入乳杆菌感受态NC8中。制备重组Gal-6基因乳酸菌。通过SDS-PAGE、Western blotting方法检测Gal-6在植物乳杆菌中的表达情况,应用薄层平皿琼脂糖扩散法测定重组乳酸菌的抑菌活性。结果显示成功构建了重组质粒pSIP409-Gal-6,并利用电转化技术成功转入植物乳酸杆菌中,通过检测验证表达成功。抑菌活性实验证明Gal-6分别对金黄色葡萄球菌和大肠杆菌具有一定的抑菌活性。为进一步分析重组乳酸菌的免疫保护性效果,验证Gal-6是否能增强鸡的机体免疫功能,增强抗病和抗感染能力,将构建的重组乳酸菌pSIP409-Gal-6经SppIP诱导表达后饲喂雏鸡,45只2-3周龄的雏鸡随机分成3组,每组各15只,三组分别为pSIP409-Gal-6组,pSIP409组和PBS组,以重组乳酸菌5.0×109 CFU分组口服免疫雏鸡,每只免疫计量控制为0.3ml,按以上计量进行免疫三周,第四周停止免疫,第五周再次进行免疫,免疫周期结束后对鸡翅下采血并用流式细胞术检测CD4+和CD8+T淋巴细胞。口服免疫结束2周后用致病性大肠杆菌ETEC 5.0×1010 CFU进行攻毒实验。攻菌后每天监测体重,检测各组实验鸡的体重变化。攻毒后第10天处死,取十二指肠、回肠、盲肠制作石蜡包埋组织切片,H-E染色后观察肠道病理变化。结果显示,pSIP409-Gal-6组和pSIP409组的体重丢失低于PBS组。从流式细胞仪检测结果看出,Gal-6能够增加CD4+T淋巴细胞数量,增强机体免疫力。攻毒后肠道病理切片得出,口服重组乳酸菌pSIP409-Gal-6能改善肠道病理变化。因此,将β-防御素通过乳酸菌在动物体内表达,增强动物机体抗感染能力,这对动物性食品安全和公共卫生具重要意义,对减少和替代抗生素的使用起到积极促进作用。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79

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