發(fā)布時(shí)間：2018-05-09 10:45

  本文選題：布魯氏菌 + 保護(hù)性抗原　； 參考：《吉林大學(xué)》2011年碩士論文

【摘要】：背景 布魯氏菌病(簡(jiǎn)稱布病)是由布魯氏菌引起的一種人畜共患傳染病,是我國(guó)法定的乙類傳染病。人感染后可產(chǎn)生發(fā)熱等一系列癥狀,從而降低生活質(zhì)量,而母畜感染后可致其不孕,流產(chǎn)等,這些嚴(yán)重影響人們的健康和社會(huì)經(jīng)濟(jì)的發(fā)展。布魯氏菌病一旦感染,不易治愈,所以疫苗免疫對(duì)布魯氏菌病的控制就顯得尤為重要。現(xiàn)在,用于布魯氏菌病的疫苗主要分為3種：減毒活疫苗、滅活全疫苗和亞單位疫苗。減毒活疫苗是目前應(yīng)用最為廣泛的布魯氏菌疫苗,可以提供較好的免疫保護(hù)力,但是它的副作用較大,存在安全問(wèn)題；滅活全疫苗只可以提供短暫的保護(hù)力；而亞單位疫苗組分清楚,容易對(duì)生產(chǎn)進(jìn)行控制,副反應(yīng)小,在應(yīng)用方面具有廣闊的前景�，F(xiàn)今,對(duì)于布魯氏菌亞單位疫苗的研究還很少,僅限于對(duì)布魯氏菌單個(gè)抗原的研究。 目的 本研究就是利用反向疫苗學(xué)的思想將分離鑒定的布魯氏菌分泌蛋白和已經(jīng)報(bào)道的可能與布魯氏菌毒力及胞內(nèi)生存相關(guān)的蛋白在大腸桿菌中表達(dá),純化,獲得一定濃度的目的蛋白。利用獲得的蛋白對(duì)免疫后小鼠脾細(xì)胞進(jìn)行體外刺激,篩選出可以誘導(dǎo)細(xì)胞免疫的抗原蛋白進(jìn)行免疫保護(hù)力的評(píng)價(jià),為布魯氏菌亞單位疫苗提供標(biāo)靶。 方法 首先,將前期實(shí)驗(yàn)構(gòu)建好的108株布魯氏菌表達(dá)株在體外誘導(dǎo)表達(dá)。根據(jù)蛋白的可溶性及表達(dá)量將目的蛋白用鎳柱親和層析法純化,獲得布魯氏菌目的蛋白,其中包括和免疫保護(hù)性相關(guān)的毒力蛋白、分泌蛋白及外膜蛋白。用最終符合濃度的103株純化好的蛋白對(duì)S19疫苗株免疫過(guò)的小鼠脾細(xì)胞進(jìn)行體外刺激。同時(shí)同樣條件下刺激PBS免疫小鼠脾細(xì)胞,作為陰性對(duì)照組。ELISA Quantikine Mouse IFN-γkit試劑盒測(cè)定細(xì)胞培養(yǎng)上清中IFN-γ的含量。如果蛋白刺激的S19免疫組小鼠脾細(xì)胞分泌的IFN-γ含量高于PBS組小鼠脾細(xì)胞的IFN-γ含量,說(shuō)明這個(gè)蛋白可以刺激小鼠產(chǎn)生細(xì)胞免疫反應(yīng)。本試驗(yàn)中共篩選出10可以誘導(dǎo)細(xì)胞免疫的蛋白,并進(jìn)一步進(jìn)行免疫保護(hù)性分析。 其中,這10個(gè)蛋白中有3個(gè)蛋白是文獻(xiàn)已經(jīng)報(bào)道的具有保護(hù)性的蛋白,所以對(duì)剩余7個(gè)蛋白進(jìn)行了免疫保護(hù)性分析。將蛋白以佐劑乳化后免疫小鼠,腹膜下免疫兩次,S19免疫組為陽(yáng)性對(duì)照,PBS免疫組為陰性對(duì)照。在免疫后不同時(shí)間分別對(duì)小鼠體液抗體效價(jià),抗體分型測(cè)定,細(xì)胞免疫進(jìn)行測(cè)定。免疫后的小鼠感染abortus 544后30天,進(jìn)行小鼠脾細(xì)胞內(nèi)菌落計(jì)數(shù)CFU。 結(jié)果 通過(guò)以上實(shí)驗(yàn),有4個(gè)蛋白可以保護(hù)小鼠抵抗布魯氏菌的感染,產(chǎn)生較好的免疫保護(hù)效果,它們分別為BMEI0357、BMEII1048.BMEI0705和BMEI1692。這四個(gè)蛋白免疫后,小鼠脾細(xì)胞的菌落計(jì)數(shù)和PBS組小鼠脾細(xì)胞的CFU有明顯差別,基本上減少了一個(gè)數(shù)量級(jí),同時(shí)這四個(gè)蛋白可以誘導(dǎo)小鼠產(chǎn)生較高的抗體反應(yīng),但是只有蛋白BMEII1048.BMEI0705可以誘導(dǎo)小鼠產(chǎn)生細(xì)胞免疫反應(yīng)。最終篩選出2個(gè)既可以誘導(dǎo)小鼠產(chǎn)生體液免疫又產(chǎn)生細(xì)胞免疫的保護(hù)性的抗原。 結(jié)論 通過(guò)優(yōu)化蛋白表達(dá)和純化的條件,使得108個(gè)布魯氏菌蛋白的可溶性表達(dá)的比例增加,并且獲得了較為統(tǒng)一的純化蛋白濃度。用ELISA方法測(cè)定蛋白刺激的小鼠脾細(xì)胞分泌的IFN-γ的含量,從而篩選出10個(gè)可以誘導(dǎo)細(xì)胞免疫的布魯氏菌的候選蛋白,3個(gè)可以抑制小鼠脾細(xì)胞產(chǎn)生細(xì)胞免疫的蛋白。在這10個(gè)可以誘導(dǎo)小鼠產(chǎn)生細(xì)胞免疫的蛋白中,除了已經(jīng)報(bào)道的具有較好免疫保護(hù)效果的蛋白外,篩選出4個(gè)具有保護(hù)效果的候選蛋白。對(duì)上述蛋白小鼠的體液免疫反應(yīng)鑒定結(jié)果可知,它們都可以誘導(dǎo)細(xì)胞產(chǎn)生特異的IgG抗體,多數(shù)抗體亞型以IgG2a亞型為主。對(duì)以上幾組小鼠的細(xì)胞免疫反應(yīng)鑒定結(jié)果顯示,BMEII1048、BMEI0705在佐劑混合條件下免疫可以刺激機(jī)體產(chǎn)生Th1細(xì)胞免疫反應(yīng),提示可以作為布魯氏菌保護(hù)性抗原的候選蛋白,為布魯氏菌亞單位疫苗提供靶標(biāo)。
[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 - 緯 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 - 緯 was measured by ELISA Quantikine Mouse IFN - 緯 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 .

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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本文編號(hào)：1865721