细菌脂多糖的制备与纯化

细菌脂多糖的提取与制备

生技052 张创峰

指导教师宫强

摘要

目的：提取制备出鸡白痢细菌的脂多糖（Lipopolysaccharides LPS），并检测出其纯度，免疫动物后检测其诱导机体产生抗体的效价。

方法：对刚接种的细菌进行37℃培养过夜，此时菌体大致处于对数期。利用热酚水法提取LPS，通过离心、反复冻融对菌体进行破碎，再透析、浓缩进行LPS粗提取，进而通过酶解、加丙酮沉淀等一系列步骤纯化LPS；然后利用紫外光谱法、标准曲线法和硫酸蒽酮法分别对制备的LPS溶液中核酸、蛋白、糖的含量进行分析测定，配制成适合注射的LPS浓度；最后通过皮下注射免疫鸡，对鸡血进行离心得到血清，保存血清。免疫三次，采血三次，利用间接Elisa法测定抗体的效价。

结果：成功提取了细菌的LPS，免疫注射浓度为121.5μg/mL。未经酶解的粗制LPS于260nm处有强吸收，经酶解处理分离纯化的LPS，于260nm处吸收值较小。经分光光度计测定，结果为0.324，从标准曲线找出其蛋白含量为0.59mg/mL。然而所提取的LPS并未使动物产生抗体，免疫动物不成功。

关键词：鸡白痢细菌，脂多糖（LPS），提取，糖含量，免疫，ELISA

Extraction and Preparation of Salmonella

Lipopolysaccharide

Grade 2005, Class2, Major of Biotechnology Chuagfeng Zhang

Tutor Qiang Gong

ABSTRACT

Objective This study was to extract and prepare Salmo nella pullorum’s Lipopolysaccharides(LPS), and test its purity, after immune the animals, we will detect its antibody titer induced by the production.

Methods Put the vaccinated Salmonella at just 37℃ overnight train, to ensure the biomass is at logarithmic phase, and then extraction of LPS from biomass with mixture of hot phenol-water, through centrifugation and repeated freezing and thawing the biomass to carry out broken the biomass, and then dialysis, dialysis the solution for the crude LPS extraction, through the zymohydrolysis, adding acetone to precipitate LPS, using the ultraviolet spectroscopy, the BSA standard curve method and anthrone-sulfuric acid method for testing the content of nucleic acids, protein, sugar of the prepared LPS solution. Finally, through the subcutaneous injection to immune chicken, to obtain serum from centrifuged blood, preservation serum, immunization three times, blood three times, select chicken serum antibodies, using an indirect ELISA method to detect the titer of antibody.

Results We successfully extracted LPS from the Salmonella, the concentration for immunization is 121.5μg/mL. Without the enzyme in the crude LPS there is strong absorption of 260nm by the enzyme to deal with separation and purification of LPS, in a relatively there is small absorption of 260nm Department. Measured by the spectrophotometer, the results is 0.324, we can find out the protein content is 0.59mg/mL from the standard curve. Nevertheless, the extraction of LPS did not make animals generate antibodies, the immune on animals is unsuccessful.

KEY WORDS: Salmonella Pullorum, Lipopolysaccharides(LPS), extracts, sugar content, immunization, ELISA

目录

前言 (1)

第1章文献综述 (2)

1.1 鸡白痢简介 (2)

1.1.1 病原 (2)

1.1.2 传播流行特点 (2)

1.1.3 病理变化 (2)

1.1.4 检测手段 (3)

1.1.5 研究趋势 (4)

1.2 LPS研究进展 (5)

1.2.1 LPS的来源 (6)

1.2.2 LPS的结构 (6)

1.2.3 LPS提取方法的研究 (6)

第2章实验材料与方法 (8)

2.1实验材料 (8)

2.1.1菌株与实验动物 (8)

2.1.2 试剂 (8)

2.1.3 主要溶液的配制 (8)

2.1.4 主要仪器 (10)

2.2 实验方法 (10)

2.2.1培养基的配制 (10)

2.2.2菌体的获得 (11)

2.2.3 LPS粗品的制备 (11)

2.2.4 LPS的纯化 (12)

2.2.5 LPS某些物质含量的测定 (12)

2.2.6 O-SP制备 (13)

2.2.7 免疫与采血 (13)

2.2.8 血清分离 (13)

2.2.9抗体效价的测定 (13)

第3章结果与讨论 (15)

3.1 结果 (15)

3.1.1 LPS核酸的检测 (15)