【作者】 魏春梅；

【导师】 彭广能； 杨宏军；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2010， 硕士

【摘要】 本试验通过采用金葡菌Newbould305建立了小鼠泌乳期乳腺内感染的乳腺炎动物模型,并在此基础上探讨牛源性重组金葡菌β-溶血素(P-hemolysin,β-HL)亚单位疫苗对小鼠乳腺局部、整体的防御体系及炎症相关因子的影响,为乳腺炎的生物防治提供新思路。1.牛源性金葡菌攻击下小鼠泌乳期感染模型的建立选择48只未孕雌鼠随机分为6组(n=8),产后7-10d分别在小鼠的第四对乳腺内灌注经过毒力活化的细菌悬液5.0×10~2CFU/50μL (E1)、1.0×10~3CFU/50μL (E2)、5.0×10~3CFU/50μL (E3)、1.0×10~4CFU/50μL (E4)和1.0×10~5CFU/50μL (E5),对照组小鼠乳腺内注入同剂量的无菌的PBS,48h处死。观察小鼠乳腺组织中细菌数量、TNF-α、IL-2和IL-6含量的变化及乳腺组织形态学观察。结果表明实验组乳腺组织中分离到细菌数呈现先升高后下降的趋势,E3达到最高。组织形态学上对照组腺泡结构完整, E1组和E2组腺泡内主要以浆液性渗出为主,E3组腺泡壁增厚,腺泡腔内嗜中性粒细胞(PMN)增多,随着剂量的增大E4和E5组腺泡壁破裂,甚至个别样品出现坏死灶。与对照组相比,E3组小鼠乳腺组织中TNF-α、IL-2和IL-6含量极显著升高(P<0.01),整体来看呈先升高后下降的趋势。由结果可知5.0×10~3CFU/50μl(E3)剂量下的金葡菌攻击下的小鼠乳腺炎性反应与炎症的免疫病理过程一致,故选择此剂量作为小鼠乳腺内感染的病理模型的造模剂量。2.重组金葡菌p-溶血素亚单位疫苗对小鼠金葡菌性乳腺炎的保护效果研究48只雌鼠随机分成对照组、造模组、pET-32a(+)质粒蛋白免疫不攻毒组、pET-32a(+)质粒蛋白免疫攻毒组、β-HL免疫不攻毒组、β-HL免疫攻毒组(n=8),用Ni-NTA Agarose纯化目的蛋白,Bradfrod法对目的蛋白进行蛋白含量测定,无菌将蛋白制作成疫苗免疫小鼠。产后7-10天分别在小鼠的第四对乳腺内灌注经过毒力活化的细菌悬液5.0×10~3CFU/50μL,用ELISA方法测定抗体效价,并分离攻毒后小鼠乳腺中细菌、观察乳腺匀浆中细胞因子的含量变化及通过琼脂糖免疫双向扩散试验观察pET-32a(+)载体对金葡菌重组β-溶血素的影响。结果表明纯化的β-HL和pET-32a (+)质粒蛋白分别在57kD和20.5kD处显示单一条带,蛋白含量分别为950μg/ml和1.45mg/ml;β-HL重组蛋白和pET-32a(+)质粒蛋白免疫小鼠都能产生相应的抗体,随着免疫次数的增加,产生的相应抗体滴度也逐渐增加；pET-32a(+)免疫攻毒组和β-HL免疫攻毒组乳腺匀浆中的TNF-α和IL-2的含量与空白攻毒组相比有所变化,但差异不显著(P>0.05),之间不存在统计学意义,但与各自相应对照组之间存在统计学意义。而pET-32a(+)免疫攻毒组乳腺匀浆中的IL-6含量与空白攻毒组之间,二者存在统计学意义(P<0.05),而p-HL免疫攻毒组则变化不大；通过琼脂糖免疫双向扩散试验,抗重组β-HL抗体和抗pET-32a(+)载体蛋白的抗体都与金葡菌Newbould305超声波破碎液形成沉淀线。由结果可知纯化的目的蛋白达到电泳级纯度；蛋白疫苗作用机体能够产生较高的抗体水平；pET-32a(+)质粒蛋白可以增加β-HL蛋白的抗原性。总之,金葡菌重组β-溶血素亚单位疫苗对由金葡菌菌株Newbould305诱导的乳腺炎并不能减轻小鼠乳腺的炎症状态,但一定程度上能够减少小鼠乳腺内细菌的数量。更多还原

【Abstract】 The mastitis model induced by Staphylococcus aureus (S.aureus) Newbould305 via the base of the mammary glands in mice was established successfully in this study. Therefore the protective effect of recombinant Staphylococcal Beta-hemolysin vaccine on immune defensive system and the inflammation-associated factors of mammary gland and systematic level by using this mastitis model were investigated. The result of this study provided a new for the biotherapy of mastitis.1. Establishment of lactation period mammary gland attacked by Staphylococcus aureus isolated from cows in mice48 mice were randomly divided into 6 groups(n=8),5.0×10~2CFU/50μL(E1)、1.0×10~3CFU/50μL(E2)、5.0×10~3CFU/50μL(E3).1.0×10~4CFU/50μL(E4) 1.0×10~5CFU/50μL(E5) and control group were inoculated into the base of the mammary glands 7-10 days after parturition. All the mice were sacrificed at 48h after infection. The content changes of mouse-interleukin 2(IL-2)、mouse-interleukin 6(IL-6)、mouse-tumor necrosis factor a(TNF-α)、histopathologic observations and the amount of bacteria from mammary glands of mice. Breast tissue in the experimental groups, the number of isolated bacteria showed a downward trend after the first rise, E3 highest. Histopathologic examination of mammary gland revealed that the acinar structural of the control group were present integrity. The El and E2 groups were present primarily within the acinus serous effusion-based, histopathologic examination of the E3 group showed that numbers of polymorphonuclear neutrophils (PMN) were increased and acinus wall thickening were thickend. As the dose increase, the acinus wall of the E4 and E5 groups ruptured, and even individual samples appeared necrotic foci. In comparison with the control group, TNF-α、IL-2 and IL-6 concentration of the mammary gland tissue of the E3 group were significantly (P< 0.01) higher and overall tend showed increased at the beginning,and then decreased. The results demonstrated that 5.0×10~3CFU/50μL (E3) under the attack of doses of the Staphylococcus aureus was the same as inflammatory response and immunopathology, so choose this dose as the mice with mammary gland infection in the pathological model of modeling dose.2. The Protective Effect of recombinant Staphylococcal Beta-hemolysin vaccine on Mice with experimental Mastitis Induced by Staphylococcus aureus Infection48 mice were randomly divided into blank control group and blank control challenged group and pET-32a (+) vaccinated without challenged group and pET-32a (+) vaccinated challenged group and P-HL vaccinated without challenged group andβ-HL vaccinated challenged group (n=8), Ni-NTA Agarose was used to purify purpose fusion protein and protein content were determined with Bradfrod method, then, the purpose protein purified were aseptically made into vaccine. All the mice of all the challenged groups were inoculated into the base of the mammary glands 7-10 days after parturition with 5.0×10~3CFU/50μL of bacterial suspension. The mammary glands were harvested and ground for calculating colony for units and viewing the content changes of the inflammation-associated factors of mammary gland. Agarose immune-double diffusion test was reviewed if pET-32a (+) has an impact against recombinant Staphylococcal Beta-hemolysin.The results showed that there were expected protein bands with molecular mass of 57kD (purifiedβ-HL) and 20.5kD (purified pET-32a (+) in SDS-PAGE, and the concentration were 950μg/ml and 1.45mg/ml in differences. The blood sera of all vaccinated mice were detected specific antibodies. Moreover, the antibody titers were present to step up with increased number of times of vaccination. The concentrations of mouse-tumor necrosis factor a(TNF-α) and mouse-interleukin 2(IL-2) in mammary gland from pET-32a (+)vaccinated challenged group and P-HL vaccinated challenged group were a degree to change for comparison with the blank control challenged group (P> 0.05), however, in comparison with corresponding to control groups, there were present in statistical significance (P< 0.05). However, The content of mouse-interleukin 6(IL-6) fromβ-HL vaccinated challenged group were only present in statistical significance (P< 0.05) for comparison with the blank control challenged group. Agarose immune-double diffusion test revealed that the holes of theantibody of purifiedβ-HL fusion protein and the antibody of purified pET-2a (+) carrier protein emerge sediment lines with that of disintegration suspension of Staphylococcus aureus (S.aureus) Newbould305.Based on these results, we investigated that the purified fusion proteins had good fineness and injected vaccine to mice can produce a higher antibody titer and the pET-32a (+) carrier can increase the antigenicity of the purifiedβ-HL fusion protein. In conclusion, the preventive efficacy of the recombinantβ-HL vaccine against infection in lactating mice inoculated with Staphylococcus aureus (S.aureus) Newbould305 couldn’t lessen inflammation symptom of mice, however, which could decrease degreely bacterial numbers in mammary gland.更多还原