本文選題：嗜水氣單胞菌 + 微囊口服疫苗�。� 參考：《南京農(nóng)業(yè)大學(xué)》2008年碩士論文

【摘要】：嗜水氣單胞菌(Aeromonas hydrophila,Ah)是淡水魚類的重要病原菌,給淡水養(yǎng)殖業(yè)帶來了巨大的經(jīng)濟(jì)損失,同時也給人類健康帶來潛在威脅。嗜水氣單胞菌可引起淡水魚類的出血性敗血癥,具有高死亡率。因此,規(guī)�；B(yǎng)殖場嗜水氣單胞菌免疫與防控尤為重要。口服免疫是規(guī)�；~類養(yǎng)殖中具有良好應(yīng)用前景的免疫方式。但是由于胃腸復(fù)雜環(huán)境對疫苗抗原成分的降解,其免疫效果往往不佳。本研究的目的是利用生物相容性好的高分子聚合物制備包裹有全菌抗原的微囊傳遞系統(tǒng),開發(fā)嗜水氣單胞菌口服疫苗。 以海藻酸鈉(Sodium alginate,SA)和殼聚糖(Chitosan)為材料,利用改良噴霧-離子交聯(lián)技術(shù)制備嗜水氣單胞菌微囊(Microsphere,MS)口服疫苗。通過對影響微囊粒徑的細(xì)菌濃度、海藻酸鈉濃度、噴霧速度及攪拌速度等主要技術(shù)參數(shù)進(jìn)行正交篩選,確定最優(yōu)的制備技術(shù)路線,并對該口服疫苗微囊的形態(tài)、直徑、包被效率、載藥量、體外模擬釋放性、抗原穩(wěn)定性、安全性、儲存穩(wěn)定性等特性進(jìn)行檢測。所確定的最優(yōu)微囊制備參數(shù)為:SA濃度2%(W/V),細(xì)菌濃度5×10~9 cfu/mL,攪拌速度800rpm,噴霧速度7mL/min;微囊圓整,平均粒徑為11.010±0.145μm,粒徑分布較均勻,83%的微囊直徑小于15μm;包被率約(96.52±0.17)%;載菌量為(2.41±0.32)×10~8 cfu/mg;微囊在模擬胃腸條件下穩(wěn)定,表現(xiàn)出較好的突釋性與緩釋性;微囊中細(xì)菌抗原性保持良好,疫苗安全性與儲存穩(wěn)定性較好。結(jié)果表明所開發(fā)的嗜水氣單胞菌微囊傳遞系統(tǒng)各項特性較理想,有進(jìn)一步開發(fā)的前景。 為了評價所研制的嗜水氣單胞菌海藻酸鈉-殼聚糖微囊(Ah-ACMS)口服疫苗在動物體內(nèi)的免疫效果,采用不同劑量、不同劑型(有無佐劑)的Ah-ACMS疫苗分別口服免疫BALB/C小鼠,對免疫小鼠的血清抗體水平、巨噬細(xì)胞吞噬活性、淋巴細(xì)胞主要細(xì)胞因子相對表達(dá)量及淋巴細(xì)胞轉(zhuǎn)化率等多個免疫指標(biāo)進(jìn)行了檢測分析,并通過攻毒試驗對疫苗的保護(hù)效率進(jìn)行了評價。初次免疫四周后,高、中、低口服疫苗免疫組的各項免疫學(xué)參數(shù)與細(xì)菌口服對照組及空白對照組均差異顯著(P＜0.05),高、中劑量口服疫苗組間大部分免疫參數(shù)差異不顯著(P＞0.05)。Ah+SA+Ch口服組與空白對照組的脾淋巴細(xì)胞主要細(xì)胞因子表達(dá)及巨噬細(xì)胞吞噬活性等免疫指標(biāo)差異顯著。各免疫組小鼠的相對保護(hù)效率分別為:ACMS高劑量組46.7%;ACMS佐劑組53.3%;Ah腹腔注射組86.7%;而空白對照組與細(xì)菌口服對照組的小鼠100%死亡。結(jié)果表明所制備微囊口服疫苗對Ah感染具有一定的免疫抵抗力,所用口服佐劑(MonTanide IMS1312)能顯著增強(qiáng)微囊口服疫苗的免疫效果,使小鼠的相對保護(hù)率提高了13.3%。 由于哺乳動物與魚類的體溫、免疫系統(tǒng)及胃腸環(huán)境等生理參數(shù)有較大差異,為了科學(xué)評價所開發(fā)微囊口服疫苗的對水生動物的免疫效果,采用不同劑量,不同劑型(佐劑有無)的Ah-ACMs疫苗分別口服免疫銀鯽,以腹腔注射福爾馬林滅活的Ah(FKC)為陽性對照。初次免疫四周后,對免疫銀鯽的血清抗體水平、巨噬細(xì)胞吞噬活性等免疫指標(biāo)進(jìn)行了檢測分析,并通過攻毒試驗對疫苗的保護(hù)效率進(jìn)行了評價。低劑量的口服Ah-ACMS組的各項免疫指標(biāo)與空白對照組之間有顯著差異(P＜0.05)。佐劑可顯著增強(qiáng)所開發(fā)微囊口服疫苗的免疫原性。低劑量Ah-ACMS組,佐劑Ah-ACMS組及腹腔注射組的相對保護(hù)率分別為:32.1%、50.0%、82.1%,而FKC口服對照組與空白對照組鯽魚死亡率分別為96.7%和93.3%。結(jié)果表明所開發(fā)的Ah-ACMS疫苗對鯽魚具有一定的免疫效應(yīng),可保護(hù)鯽魚抵抗嗜水氣單胞菌的感染。 綜上所述,本研究所開發(fā)的聚合物微囊抗原傳遞系統(tǒng)可有效保護(hù)全菌抗原的免疫原性,對復(fù)雜的胃腸環(huán)境耐受,可誘導(dǎo)小鼠及鯽魚產(chǎn)生免疫應(yīng)答。該研究以期能為后繼的聚合物微囊口服疫苗的開發(fā)提供參考。有望進(jìn)一步推廣應(yīng)用于其他魚類病原菌口服疫苗的開發(fā)。
[Abstract]:Aeromonas hydrophila (Ah), an important pathogen of freshwater fishes, has brought huge economic losses to the freshwater aquaculture industry and also poses a potential threat to human health. Aeromonas hydrophila can cause hemorrhagic septicemia in freshwater fishes and have high death and death rates. Therefore, the immunization of Aeromonas hydrophila in large-scale farms. And prevention and control is particularly important. Oral immunization is a promising application of immunization in large-scale fish culture. However, the immune effect is often poor due to the degradation of antigen components in the complex gastrointestinal environment. The purpose of this study is to prepare microcapsules encapsulated with full bacterial antigen by good biocompatible polymer polymer. An oral vaccine for Aeromonas hydrophila was developed.
Taking sodium alginate (Sodium alginate, SA) and chitosan (Chitosan) as materials, the oral vaccine of Aeromonas hydrophila microcapsule (Microsphere, MS) was prepared by improved spray ion crosslinking technology. The main technical parameters, such as the concentration of bacteria, the concentration of alginate, the velocity of spray and the stirring speed, were screened by the orthogonal screening of the bacteria concentration affecting the microcapsule diameter. The optimal preparation technique was used to detect the morphology, diameter, encapsulation efficiency, drug loading, in vitro analogue release, antigen stability, safety and storage stability of the oral vaccine. The optimum microcapsule preparation parameters were SA concentration 2% (W/V), bacterial concentration 5 x 10~9 cfu/mL, stirring speed 800rpm, spray velocity 7mL/m In, the microcapsules were round and round, the average particle size was 11.010 + 0.145 mu m, the particle size distribution was more uniform, the diameter of the microcapsule was less than 15 mu m, the inclusion rate was about (96.52 + 0.17)%, and the carrier amount was (2.41 + 0.32) x 10~8 cfu/mg, and the microcapsules were stable under simulated gastrointestinal conditions and showed good release and sustained release; the bacteria antigenicity in microcapsules was good and vaccine safety and vaccine safety The storage stability was good. The results showed that the Aeromonas hydrophila microcapsule delivery system had better characteristics and had further development prospects.
In order to evaluate the immune effect of the oral vaccine of Aeromonas hydrophila sodium alginate chitosan microcapsule (Ah-ACMS) in animals, the Ah-ACMS vaccine of different doses and different dosage forms (without adjuvant) was used to immunize BALB/C mice orally. The anti body level of the serum, the phagocytic activity of macrophage and the main lymphocyte of the mice were mainly fine. The relative expression of cytokine and lymphocyte transformation rate were detected and analyzed, and the protective efficiency of the vaccine was evaluated by the attack test. After the first four weeks, the immunological parameters of the high, middle and low oral immunization groups were significantly different from those in the oral control group and the blank control group (P < 0.05). The difference of most of the immune parameters between high and middle doses was not significant (P > 0.05) in the.Ah+SA+Ch oral group and the blank control group, the expression of main cytokines and the phagocytic activity of macrophages in the control group were significantly different. The relative protective efficiency of the mice in each immunization group was 46.7% in ACMS high dose group and 53.3% in the ACMS adjuvant group. The Ah intraperitoneal injection group was 86.7%, while the mice in the blank control group and the bacterial oral control group died in 100%. The results showed that the oral vaccine prepared by the prepared microcapsules had certain immune resistance to Ah infection, and the oral adjuvant (MonTanide IMS1312) could significantly enhance the immunization effect of the oral vaccine of microcapsules and increase the relative protection rate of the mice by 13.3%..
In order to evaluate the immune effect of the microcapsule oral vaccine for aquatic animals, the Ah-ACMs vaccine of different doses and different dosage forms (adjuvant or not) was taken orally to the inactivated Ah of the formalin in order to evaluate the immune effect of the microcapsule oral vaccine. FKC) was a positive control. After the first four weeks of immunization, the serum antibody level of the silver crucian carp and the phagocytic activity of macrophage were detected and analyzed. The protective efficiency of the vaccine was evaluated by the attack test. There was a significant difference between the immunization index and the blank control group in the low dose group of oral Ah-ACMS group (P < 0.05). The relative protection rate of the low dose Ah-ACMS group, the adjuvant Ah-ACMS group and the intraperitoneal injection group were 32.1%, 50%, 82.1%, respectively, while the mortality rate of the FKC oral control group and the blank control group was 96.7% and 93.3%. respectively, indicating that the Ah-ACMS vaccine developed by the Ah-ACMS was one of the crucian carp. The immunological effect can protect crucian carp against infection by Aeromonas hydrophila.
To sum up, the polymer microcapsule antigen transfer system developed in this study can effectively protect the immunogenicity of all bacterial antigens and can induce immune responses in mice and Carassius auratus in complex gastrointestinal environments. This study is expected to provide a reference for the development of subsequent oral vaccine for microcapsules of polymer microcapsules. Development of oral vaccine for the pathogenic bacteria of fish.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳光榮,肖克宇,鄧時銘;魚用疫苗基因工程技術(shù)及其商品化初探[J];北京水產(chǎn);2003年03期

2 朱芝秀,曾志明,何后軍,鄧舜洲,鄔向東,王萍,李暉;嗜水氣單胞菌滅活苗的研制[J];江西農(nóng)業(yè)大學(xué)學(xué)報;2003年05期

3 陳懷青,陸承平;家養(yǎng)鯉科魚暴發(fā)性傳染病的病原研究[J];南京農(nóng)業(yè)大學(xué)學(xué)報;1991年04期

4 楊先樂,夏春,左文功,吳泉明,郭永金;草魚出血病細(xì)胞培養(yǎng)滅活疫苗的研究——生產(chǎn)性免疫試驗[J];水產(chǎn)科技情報;1992年01期

5 陸承平;致病性嗜水氣單胞菌及其所致魚病綜述[J];水產(chǎn)學(xué)報;1992年03期

6 儲衛(wèi)華,陸承平;PCR擴(kuò)增特異性16SrDNA和溶血素基因檢測致病性嗜水氣單胞菌[J];水產(chǎn)學(xué)報;2005年01期

7 單曉楓,高云航,李影,錢愛東;魚用疫苗的研究[J];中國水產(chǎn);2005年07期

8 劉善奎,鐘延強(qiáng),王芳,孫樹漢,高申;口蹄疫DNA疫苗海藻酸鈉微球的制備及體外釋放的研究[J];藥學(xué)服務(wù)與研究;2004年02期

9 張吉紅,儲衛(wèi)華,陸承平;口服嗜水氣單胞菌生物被膜疫苗的動物免疫試驗[J];中國水產(chǎn)科學(xué);2004年05期

10 秦磊,姚火春,陸承平;嗜水氣單胞菌與遲緩愛德華氏菌亞單位二聯(lián)疫苗對小鼠的免疫效果[J];中國獸醫(yī)學(xué)報;2000年02期

，

本文編號：2073026