布鲁氏菌保护性抗原的筛选和初步评价

发布时间：2018-05-09 10:45

本文选题：布鲁氏菌 + 保护性抗原　；参考：《吉林大学》2011年硕士论文

【摘要】：背景布鲁氏菌病(简称布病)是由布鲁氏菌引起的一种人畜共患传染病,是我国法定的乙类传染病。人感染后可产生发热等一系列症状,从而降低生活质量,而母畜感染后可致其不孕,流产等,这些严重影响人们的健康和社会经济的发展。布鲁氏菌病一旦感染,不易治愈,所以疫苗免疫对布鲁氏菌病的控制就显得尤为重要。现在,用于布鲁氏菌病的疫苗主要分为3种：减毒活疫苗、灭活全疫苗和亚单位疫苗。减毒活疫苗是目前应用最为广泛的布鲁氏菌疫苗,可以提供较好的免疫保护力,但是它的副作用较大,存在安全问题；灭活全疫苗只可以提供短暂的保护力；而亚单位疫苗组分清楚,容易对生产进行控制,副反应小,在应用方面具有广阔的前景。现今,对于布鲁氏菌亚单位疫苗的研究还很少,仅限于对布鲁氏菌单个抗原的研究。目的本研究就是利用反向疫苗学的思想将分离鉴定的布鲁氏菌分泌蛋白和已经报道的可能与布鲁氏菌毒力及胞内生存相关的蛋白在大肠杆菌中表达,纯化,获得一定浓度的目的蛋白。利用获得的蛋白对免疫后小鼠脾细胞进行体外刺激,筛选出可以诱导细胞免疫的抗原蛋白进行免疫保护力的评价,为布鲁氏菌亚单位疫苗提供标靶。方法首先,将前期实验构建好的108株布鲁氏菌表达株在体外诱导表达。根据蛋白的可溶性及表达量将目的蛋白用镍柱亲和层析法纯化,获得布鲁氏菌目的蛋白,其中包括和免疫保护性相关的毒力蛋白、分泌蛋白及外膜蛋白。用最终符合浓度的103株纯化好的蛋白对S19疫苗株免疫过的小鼠脾细胞进行体外刺激。同时同样条件下刺激PBS免疫小鼠脾细胞,作为阴性对照组。ELISA Quantikine Mouse IFN-γkit试剂盒测定细胞培养上清中IFN-γ的含量。如果蛋白刺激的S19免疫组小鼠脾细胞分泌的IFN-γ含量高于PBS组小鼠脾细胞的IFN-γ含量,说明这个蛋白可以刺激小鼠产生细胞免疫反应。本试验中共筛选出10可以诱导细胞免疫的蛋白,并进一步进行免疫保护性分析。其中,这10个蛋白中有3个蛋白是文献已经报道的具有保护性的蛋白,所以对剩余7个蛋白进行了免疫保护性分析。将蛋白以佐剂乳化后免疫小鼠,腹膜下免疫两次,S19免疫组为阳性对照,PBS免疫组为阴性对照。在免疫后不同时间分别对小鼠体液抗体效价,抗体分型测定,细胞免疫进行测定。免疫后的小鼠感染abortus 544后30天,进行小鼠脾细胞内菌落计数CFU。结果通过以上实验,有4个蛋白可以保护小鼠抵抗布鲁氏菌的感染,产生较好的免疫保护效果,它们分别为BMEI0357、BMEII1048.BMEI0705和BMEI1692。这四个蛋白免疫后,小鼠脾细胞的菌落计数和PBS组小鼠脾细胞的CFU有明显差别,基本上减少了一个数量级,同时这四个蛋白可以诱导小鼠产生较高的抗体反应,但是只有蛋白BMEII1048.BMEI0705可以诱导小鼠产生细胞免疫反应。最终筛选出2个既可以诱导小鼠产生体液免疫又产生细胞免疫的保护性的抗原。结论通过优化蛋白表达和纯化的条件,使得108个布鲁氏菌蛋白的可溶性表达的比例增加,并且获得了较为统一的纯化蛋白浓度。用ELISA方法测定蛋白刺激的小鼠脾细胞分泌的IFN-γ的含量,从而筛选出10个可以诱导细胞免疫的布鲁氏菌的候选蛋白,3个可以抑制小鼠脾细胞产生细胞免疫的蛋白。在这10个可以诱导小鼠产生细胞免疫的蛋白中,除了已经报道的具有较好免疫保护效果的蛋白外,筛选出4个具有保护效果的候选蛋白。对上述蛋白小鼠的体液免疫反应鉴定结果可知,它们都可以诱导细胞产生特异的IgG抗体,多数抗体亚型以IgG2a亚型为主。对以上几组小鼠的细胞免疫反应鉴定结果显示,BMEII1048、BMEI0705在佐剂混合条件下免疫可以刺激机体产生Th1细胞免疫反应,提示可以作为布鲁氏菌保护性抗原的候选蛋白,为布鲁氏菌亚单位疫苗提供靶标。
[Abstract]:Background

Brucea is a kind of zoonotic infectious disease caused by brucella . It is a legal class B infectious disease in China . It can produce a series of symptoms such as fever after infection , which seriously affects the development of people ' s health and social economy .
the inactivated whole vaccine can only provide short protection force ;
However , the subunit vaccine component is clear , easy to control the production , has little side reaction , has a broad prospect in the application . Nowadays , the research on the brucella subunit vaccine is very rare , which is limited to the study of the single antigen of brucella .

Purpose

In this study , we use the idea of reverse vaccine to express and purify the isolated brucella secretion protein and the protein which has been reported possibly related to the virulence and intracellular survival of brucella , and purify to obtain a certain concentration of the target protein . The obtained protein is used to stimulate the spleen cells of the immunized mice in vitro , so as to screen out the evaluation of the immune protection force of the antigen protein which can induce cellular immunity , and provide a target for the brucella subunit vaccine .

method

The expression of IFN - 纬 in spleen cells of mice immunized with S19 vaccine strain was determined by means of affinity chromatography with nickel column , and the content of IFN - 纬 was measured by ELISA Quantikine Mouse IFN - 纬 kit .

Among them , three of the 10 proteins were protective proteins which were reported in the literature , so the remaining 7 proteins were subjected to immune protective analysis . After emulsified with adjuvant , the mice were immunized twice , the S19 immunization group was positive control group , and the PBS group was negative control . After immunization , the antibody titer , antibody typing and cellular immunity of mice were measured . After the immunization , the mice were infected by abortus 544 for 30 days , and the CFU of CFU was counted in the spleen cells of mice .

Results

The results showed that four proteins could protect mice against brucella infection and produce better immune protective effect , which were BMEI0357 , BMEII104.BMEI0705 and BMEI1692 , respectively .

Conclusion

By optimizing the expression and purification conditions of the protein , the soluble expression of 108 brucella proteins was increased , and a more uniform concentration of purified protein was obtained . Four candidate proteins with protective effect were screened by ELISA .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【参考文献】