非致病性大腸桿菌鞭毛蛋白對O型口蹄疫病毒的佐劑效果
發(fā)布時(shí)間：2018-11-15 11:21
【摘要】：【目的】口蹄疫(foot and mouth disease,FMD)的傳染性強(qiáng),傳播廣,給世界經(jīng)濟(jì)和社會發(fā)展帶來巨大危害,目前疫苗接種仍是主要而有效的防控手段。以增強(qiáng)口蹄疫疫苗免疫效力為目的,基于國內(nèi)外對鞭毛蛋白佐劑的深入了解與研究,對非致病性大腸桿菌鞭毛進(jìn)行修飾,研究鞭毛蛋白(flagellin,F)以及有無LTMT佐劑不同重組鞭毛蛋白對口蹄疫病毒(foot and mouth disease virse,FMDV)的佐劑作用�！痉椒ā客ㄟ^體外表達(dá)獲得pCold-F0、pCold-F0-LTMT、pCold-F0NC、pCold-F0NC-LTMT 4種不同重組鞭毛蛋白,用間接ELISA方法檢測pCold-F0-LTMT和pCold-F0NC-LTMT蛋白的生物學(xué)活性,按照5μg/只小鼠蛋白量與FMDV滅活抗原混合制備疫苗,同時(shí)設(shè)置添加或者不添加ISA 206佐劑組,皮下接種Balb/c小鼠,共免疫2次,每次間隔2周。分別于免疫前和免疫后14、21、28、35和45 d采血并采集小鼠糞便,45 d后取小腸后段,采用阻斷ELISA方法檢測血清IgG抗體滴度,用間接ELISA方法檢測小鼠糞便和腸洗液中特異的分泌性IgA(secretory IgA,SIgA)抗體滴度,以評估免疫效力。【結(jié)果】SDS-PAGE和Western blot分析表明,4種不同重組鞭毛蛋白成功表達(dá)。并用GM l-ELISA方法驗(yàn)證了pCold-F0-LTMT和pCold-F0NC-LTMT的生物學(xué)活性,融合蛋白F0-LTMT和F0NC-LTMT保留了與GM l結(jié)合能力。動物試驗(yàn)結(jié)果表明,疫苗經(jīng)皮下注射接種后,單獨(dú)口蹄疫抗原組沒有產(chǎn)生明顯的抗體水平,而鞭毛蛋白佐劑組比單獨(dú)的口蹄疫抗原組,IgG滴度高,并且產(chǎn)生sIgA抗體滴度更早、更高。此外,LTMT與鞭毛蛋白協(xié)同作用能刺激小鼠機(jī)體產(chǎn)生更高的IgG和sIgA。在試驗(yàn)中,當(dāng)鞭毛蛋白與ISA 206佐劑聯(lián)合使用時(shí),血清中IgG滴度大幅增高,且抗體持續(xù)期延長�？谔阋呖乖cISA 206佐劑和F0NC-LTMT混合使用效果最佳,顯示對FMDV的最好保護(hù)�！窘Y(jié)論】數(shù)據(jù)表明,黏膜佐劑F0-LTMT發(fā)揮雙重佐劑活性,皮下注射可顯著提高FMDV全身特異性IgG和局部sIgA水平,顯示其具有良好的應(yīng)用前景。
[Abstract]:[objective] Foot-and-mouth disease (foot and mouth disease,FMD) is highly infectious and widely spread, which brings great harm to the world economy and social development. At present, vaccination is still the main and effective means of prevention and control. In order to enhance the immune effectiveness of foot-and-mouth disease vaccine, based on the deep understanding and research of flagellin adjuvant at home and abroad, the flagellum of non-pathogenic Escherichia coli was modified, and the flagellin (flagellin,) was studied. F) and different recombinant flagellins with or without LTMT adjuvant for FMDV (foot and mouth disease virse,FMDV. [methods] pCold-F0,pCold-F0-LTMT,pCold-F0NC, was obtained by expression in vitro. The biological activities of pCold-F0-LTMT and pCold-F0NC-LTMT proteins were detected by indirect ELISA method. The vaccine was prepared according to 5 渭 g / mouse protein and FMDV inactivated antigen. At the same time, the Balb/c mice were inoculated subcutaneously with or without ISA 206 adjuvant. The mice were immunized twice every time for 2 weeks. The blood samples were collected before and after immunization, and the feces of mice were collected on day 14, 21, 2835 and 45, respectively. After 45 days, the posterior segment of small intestine was taken, and the titer of serum IgG antibody was detected by blocking ELISA method. Indirect ELISA assay was used to detect the specific secretory IgA (secretory IgA,SIgA antibody titers in mouse feces and intestinal lotions in order to evaluate the immune potency. [results] SDS-PAGE and Western blot analysis showed that four different recombinant flagellins were successfully expressed. The biological activities of pCold-F0-LTMT and pCold-F0NC-LTMT were verified by GM l-ELISA method. The fusion proteins F0-LTMT and F0NC-LTMT retained the ability to bind to GM l. The results of animal experiment showed that the IgG titer of FMD antigen group was higher than that of FMD antigen group, and the titer of sIgA antibody was earlier in FMD antigen group than in FMD antigen group. Higher. In addition, the synergistic effect of LTMT and flagellin could stimulate the production of higher IgG and sIgA. in mice. In the experiment, when the flagellin was combined with ISA 206 adjuvant, the serum IgG titer was significantly increased and the antibody duration was prolonged. Foot-and-mouth disease antigen combined with ISA 206 adjuvant and F0NC-LTMT showed the best protection to FMDV. [conclusion] the data showed that the mucosal adjuvant F0-LTMT had double adjuvant activity. Subcutaneous injection can significantly improve the level of systemic specific IgG and local sIgA of FMDV, which shows that it has a good application prospect.
【作者單位】： 江蘇省農(nóng)業(yè)科學(xué)院獸醫(yī)研究所/農(nóng)業(yè)部獸用生物制品工程技術(shù)重點(diǎn)實(shí)驗(yàn)室;吉林農(nóng)業(yè)大學(xué)動物科學(xué)技術(shù)學(xué)院;
【基金】：江蘇省自主創(chuàng)新資金(cx(15)1060) 國家自然科學(xué)基金(31572503)
【分類號】：S855.3
[Abstract]:[objective] Foot-and-mouth disease (foot and mouth disease,FMD) is highly infectious and widely spread, which brings great harm to the world economy and social development. At present, vaccination is still the main and effective means of prevention and control. In order to enhance the immune effectiveness of foot-and-mouth disease vaccine, based on the deep understanding and research of flagellin adjuvant at home and abroad, the flagellum of non-pathogenic Escherichia coli was modified, and the flagellin (flagellin,) was studied. F) and different recombinant flagellins with or without LTMT adjuvant for FMDV (foot and mouth disease virse,FMDV. [methods] pCold-F0,pCold-F0-LTMT,pCold-F0NC, was obtained by expression in vitro. The biological activities of pCold-F0-LTMT and pCold-F0NC-LTMT proteins were detected by indirect ELISA method. The vaccine was prepared according to 5 渭 g / mouse protein and FMDV inactivated antigen. At the same time, the Balb/c mice were inoculated subcutaneously with or without ISA 206 adjuvant. The mice were immunized twice every time for 2 weeks. The blood samples were collected before and after immunization, and the feces of mice were collected on day 14, 21, 2835 and 45, respectively. After 45 days, the posterior segment of small intestine was taken, and the titer of serum IgG antibody was detected by blocking ELISA method. Indirect ELISA assay was used to detect the specific secretory IgA (secretory IgA,SIgA antibody titers in mouse feces and intestinal lotions in order to evaluate the immune potency. [results] SDS-PAGE and Western blot analysis showed that four different recombinant flagellins were successfully expressed. The biological activities of pCold-F0-LTMT and pCold-F0NC-LTMT were verified by GM l-ELISA method. The fusion proteins F0-LTMT and F0NC-LTMT retained the ability to bind to GM l. The results of animal experiment showed that the IgG titer of FMD antigen group was higher than that of FMD antigen group, and the titer of sIgA antibody was earlier in FMD antigen group than in FMD antigen group. Higher. In addition, the synergistic effect of LTMT and flagellin could stimulate the production of higher IgG and sIgA. in mice. In the experiment, when the flagellin was combined with ISA 206 adjuvant, the serum IgG titer was significantly increased and the antibody duration was prolonged. Foot-and-mouth disease antigen combined with ISA 206 adjuvant and F0NC-LTMT showed the best protection to FMDV. [conclusion] the data showed that the mucosal adjuvant F0-LTMT had double adjuvant activity. Subcutaneous injection can significantly improve the level of systemic specific IgG and local sIgA of FMDV, which shows that it has a good application prospect.
【作者單位】： 江蘇省農(nóng)業(yè)科學(xué)院獸醫(yī)研究所/農(nóng)業(yè)部獸用生物制品工程技術(shù)重點(diǎn)實(shí)驗(yàn)室;吉林農(nóng)業(yè)大學(xué)動物科學(xué)技術(shù)學(xué)院;
【基金】：江蘇省自主創(chuàng)新資金(cx(15)1060) 國家自然科學(xué)基金(31572503)
【分類號】：S855.3
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