【摘要】：布氏桿菌(Brucella Blues)是革蘭氏陰性需氧桿菌，可引起人畜共患的布氏桿菌病，給世界各國畜牧業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失，同時(shí)也給人類健康造成極大的危害。該病目前尚不能完全根治，所以預(yù)防和檢測顯得尤為重要。 本研究采用改良熱酚法制備純化布氏桿菌M5株的脂多糖（LPS），檢測細(xì)菌LPS免疫原性；純化的細(xì)菌LPS免疫BALB/c小鼠，，通過DOT-ELISA測定血清抗體效價(jià)。結(jié)果表明采用熱酚法能獲得較高純度的細(xì)菌LPS，其免疫原性較高，DOT-ELISA檢測的血清效價(jià)達(dá)到1:106，與全菌免疫原性接近。 在此基礎(chǔ)之上，將LPS免疫的BALB/c小鼠脾細(xì)胞和SP2/0細(xì)胞進(jìn)行融合，制備雜交瘤細(xì)胞，采用間接ELISA法進(jìn)行陽性篩選，經(jīng)過三次篩選和3-4次細(xì)胞克隆化培養(yǎng)，獲得四株穩(wěn)定分泌抗體的雜交瘤細(xì)胞株，采用間接ELISA法分別測定其細(xì)胞上清抗體效價(jià)，分別達(dá)到1:104，1:105，1:105，1:105。對四株細(xì)胞的上清液進(jìn)行Tricine-SDS-PAGE鑒定，上清中同時(shí)出現(xiàn)了細(xì)胞培養(yǎng)液中沒有的蛋白，即為單克隆抗體目的蛋白。測定單克隆抗體的相對親和力和穩(wěn)定性，結(jié)果顯示其相對親和力分別為24.13μg/mL，24.65μg/mL，24.95μg/mL，23.16μg/mL，且具有良好的穩(wěn)定性。對細(xì)胞上清采用硫酸銨鹽析法初步純化，再采用親和層析法對單抗進(jìn)一步純化，純化的蛋白進(jìn)行效價(jià)測定和Tricine-SDS-PAGE鑒定，結(jié)果表明純化后四株細(xì)胞的單抗效價(jià)略有降低，但仍可達(dá)1:104。電泳結(jié)果表明單抗的輕鏈分子量約為26ku，重鏈分子量約為37ku。本實(shí)驗(yàn)結(jié)果為布氏桿菌病檢測方法的建立奠定了研究基礎(chǔ)。
[Abstract]:Brucellosis (Brucella Blues) is a gram-negative aerobic bacillus, which can cause zoonotic brucellosis, which has caused serious economic loss to animal husbandry in the world, and also caused great harm to human health. At present, the disease can not be completely cured, so prevention and detection is particularly important. In this study, the lipopolysaccharide (LPS),) of purified brucellosis M5 strain was prepared by modified thermophenol method to detect the immunogenicity of bacterial LPS, and the purified bacterial LPS was used to immunize BALB/c mice. Serum antibody titers were determined by DOT-ELISA. The results showed that the high purity bacterial LPS, could be obtained by pyrophenol method. The immunogenicity of the serum detected by DOT-ELISA was 1: 106, which was close to the immunogenicity of the whole bacterium. On this basis, the spleen cells and SP2/0 cells of BALB/c mice immunized with LPS were fused and hybridoma cells were prepared. The hybridoma cells were screened by indirect ELISA method. Four hybridoma cell lines stably secreting antibodies were obtained and their supernatant antibody titers were determined by indirect ELISA assay. The titers of the supernatant antibodies reached 1: 104 and 1: 105 respectively. The supernatant of four cell lines was identified by Tricine-SDS-PAGE, and the protein which was not found in the cell culture medium was found in the supernatant, that is, the target protein of monoclonal antibody. The relative affinity and stability of monoclonal antibody were determined. The results showed that the relative affinity of McAb was 24.13 渭 g / mL ~ (-1) 24.65 渭 g / mL ~ (-1) ~ 24.95 渭 g / m ~ (-1) 路m ~ (-1) ~ (2) 渭 g / m ~ (-1), and it had good stability. The supernatant was purified by ammonium sulfate salting-out method, and further purified by affinity chromatography. The purified protein was determined by titer and Tricine-SDS-PAGE. The results showed that the titer of the McAb was slightly decreased, but it could reach 1: 104 after purification. The results showed that the molecular weight of light chain and heavy chain were about 26kuand 37ku. respectively. The results laid a foundation for the establishment of detection method for brucellosis.
【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R392

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