本文選題：眼鏡蛇 + 蝰蛇��； 參考：《廣州醫(yī)學(xué)院》2009年碩士論文

【摘要】： 背景:抗蛇毒抗體根據(jù)來(lái)源可分為抗蛇毒血清(antivenene,AV)及抗蛇毒雞卵黃抗體(immunoglobulins in yolk, IgY)兩種,前者從哺乳動(dòng)物的血液中分離而得,后者系從禽類(lèi)的卵黃中提取。自20世紀(jì)90年代以來(lái),國(guó)外學(xué)者已從雞卵黃中制備出了抗cobra、Russell's viper、Saw-scaled viper,B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca和N. mossambica等數(shù)種IgY,并能有效中和相應(yīng)蛇毒[1,2,3]。在國(guó)內(nèi),抗眼鏡王蛇[4]、抗蝮蛇[5]、抗眼鏡蛇[6,7]和抗蝰蛇毒IgY[8]的制備及其特性研究亦有報(bào)道。與從其他哺乳動(dòng)物(如鼠、兔、豬、馬、牛等)血液中獲得抗體相比,卵黃抗體具有無(wú)法比擬的優(yōu)越性[9,10](Schade et al.1992; Larsson et al.,1991):不與蛋白G或蛋白A結(jié)合,不會(huì)激活補(bǔ)體系統(tǒng),不與類(lèi)風(fēng)濕因子結(jié)合,避免了免疫檢測(cè)中的假陰性和假陽(yáng)性問(wèn)題;雞蛋來(lái)源方便,生產(chǎn)成本低,適于大規(guī)模生產(chǎn);由免疫母雞所生的雞蛋黃中只有一種抗體,均一性好,理化性質(zhì)穩(wěn)定,耐酸,耐熱,易于保存,運(yùn)輸和使用均很方便。另外雞與哺乳動(dòng)物種系關(guān)系遠(yuǎn),少量刺激時(shí)即容易產(chǎn)生強(qiáng)免疫反應(yīng)。研制一種針對(duì)華南地區(qū)多種劇毒蛇的通用蛇傷防、治特效制劑—多價(jià)AV,可在無(wú)需明確蛇傷的毒蛇種類(lèi)情況下立即使用,以贏得有效的最佳治療時(shí)機(jī),對(duì)避免嚴(yán)重病變的發(fā)生和降低死亡率有著重要作用。在本實(shí)驗(yàn)室先前制備有單價(jià)抗眼鏡蛇及抗蝰蛇毒IgY,本實(shí)驗(yàn)擬進(jìn)一步研制雙價(jià)抗蛇毒雞卵黃抗體,為蛇傷救助提供新的途徑。 目的:通過(guò)制備雙價(jià)抗眼鏡蛇、蝰蛇毒雞卵黃抗體,檢測(cè)其生物活性,并觀察其以口服制劑的形式對(duì)眼鏡蛇、蝰蛇傷小鼠的保護(hù)作用,為蛇傷的預(yù)防和治療提供一種新的方法,為替代馬源性抗蛇毒血清奠定基礎(chǔ)。 方法: 一、雙價(jià)抗蛇毒IgY的制備與提取 1、雙價(jià)抗蛇毒IgY的制備制訂合適的免疫方案,利用本所專(zhuān)利——蛇毒取毒器制備抗原眼鏡蛇、蝰蛇毒,分別與福氏佐劑按1:1(V/V)置自制的乳化器充分乳化,初次免疫采用福氏完全佐劑乳化蛇毒,以后均用福氏不完全佐劑乳化蛇毒。每種抗原在初次免疫后的第三周、第五周各進(jìn)行1次加強(qiáng)免疫,免疫劑量間歇、逐漸加量。兩種單一抗原按順序依次交替注入單只雞體內(nèi)(雙翼、胸、腹部和背部皮下及肌肉多位點(diǎn)注射),注入間隔周期為一周。 2、雙價(jià)抗蛇毒IgY的檢測(cè)雙向免疫擴(kuò)散和免疫電泳鑒定卵黃中的特異性抗體;SDS-PAGE測(cè)定抗體產(chǎn)物的純度;以間接ELISA法檢測(cè)特異性IgY抗體效價(jià)的動(dòng)態(tài)變化以及IgY在血漿內(nèi)的效價(jià)變化。 3、雙價(jià)抗蛇毒IgY的提取采用本實(shí)驗(yàn)過(guò)程中發(fā)明的卵黃分離收集器從卵清中分離出卵黃,用水稀釋法提取IgY抗體。 二、雙價(jià)抗蛇毒IgY的生物特性研究 1、利用雙向免疫擴(kuò)散法測(cè)定抗蛇毒IgY與中國(guó)常見(jiàn)10種蛇毒的交叉免疫反應(yīng)及其反應(yīng)強(qiáng)度。2、眼鏡蛇、蝰蛇毒對(duì)卵黃膜均有不同程度的溶膜作用,用自制的卵黃膜溶膜檢測(cè)儀觀察雙價(jià)抗蛇毒IgY對(duì)蛇毒溶膜活性的中和作用。 3、雙價(jià)抗蛇毒IgY分別與眼鏡蛇、蝰蛇毒在體外中和,腹腔注射小鼠,觀察雙價(jià)抗蛇毒IgY對(duì)蛇毒致死活性的中和作用。 三、雙價(jià)抗蛇毒IgY的相關(guān)動(dòng)物實(shí)驗(yàn) 1、雙價(jià)抗蛇毒IgY按高、中、低三個(gè)濃度灌胃小鼠,在不同時(shí)間點(diǎn)殺鼠取血分離血漿,用間接ELISA法檢測(cè)灌胃小鼠血漿中雙價(jià)抗蛇毒IgY的效價(jià)。 2、雙價(jià)抗蛇毒IgY按高、中、低三個(gè)濃度灌胃小鼠,在不同時(shí)間點(diǎn)分別為小鼠腹腔注射眼鏡蛇、蝰蛇毒,檢測(cè)口服制劑雙價(jià)抗蛇毒IgY對(duì)小鼠的保護(hù)作用。 四、統(tǒng)計(jì)采用t檢驗(yàn),SPSS軟件等統(tǒng)計(jì)分析實(shí)驗(yàn)數(shù)據(jù)。 結(jié)果: 1、利用蛇毒取毒器制備的蛇毒活性高,含雜質(zhì)量少。用眼鏡蛇、蝰蛇毒粗毒、 初次小劑量免疫后,卵黃中第9天即可檢測(cè)到特異性卵黃抗體。經(jīng)間歇、逐漸加量加強(qiáng)免疫,抗體效價(jià)持續(xù)增高,達(dá)到較高水平后,能在較長(zhǎng)時(shí)間內(nèi)一直維持高效價(jià)。 2、在實(shí)驗(yàn)過(guò)程中制備了卵黃分離收集器(專(zhuān)利號(hào):2006200559807.3),利用此儀器收集試驗(yàn)用卵黃,卵清、卵黃分離度高,卵黃膜光潔、完整,卵清殘留量少,有利于進(jìn)一步的分離純化。 3、水稀釋法去脂效果好,若進(jìn)一步經(jīng)硫酸銨鹽析后,蛋白回收率約為40%。如能進(jìn)一步優(yōu)化條件,經(jīng)嗜硫色譜、疏水色譜及凝膠層析等方法純化抗體,有望得到純度更高的抗體。 4、經(jīng)雙向免疫擴(kuò)散法測(cè)定,所制雙價(jià)抗蛇毒IgY與舟山眼鏡蛇、眼鏡王蛇反應(yīng)最明顯,反應(yīng)沉淀線最清晰,與眼鏡蛇亞科和蝰蛇亞科的部分蛇毒有強(qiáng)弱不等的交叉反應(yīng);與蝮蛇亞科的蝮蛇、尖吻蝮等部分蛇毒無(wú)明顯交叉反應(yīng)。 5、根據(jù)卵黃膜溶膜檢測(cè)儀的時(shí)間結(jié)果證明,一定量的水提物抗蛇毒IgY完全能對(duì)抗眼鏡蛇、蝰蛇毒對(duì)卵黃膜的溶膜作用。 6、體外中和實(shí)驗(yàn)表明,一定濃度的水提物抗蛇毒IgY可以完全保護(hù)實(shí)驗(yàn)小鼠免受2倍LD50眼鏡蛇、蝰蛇毒的攻擊。 7、不同濃度劑量的抗蛇毒IgY能對(duì)眼鏡蛇、蝰蛇傷小鼠有不同程度的保護(hù)作用;當(dāng)IgY劑量足夠高時(shí),能夠完全保護(hù)小鼠免受眼鏡蛇、蝰蛇毒的攻擊。結(jié)論: 1、以眼鏡蛇、蝰蛇毒粗毒免疫萊杭母雞,經(jīng)初次小劑量、間歇、逐漸加量加強(qiáng)免疫,可持續(xù)數(shù)周獲得含有高效價(jià)、特異性抗體的雞卵黃抗體。 2、自制的卵黃分離收集器可批量分離卵黃,卵黃膜溶膜檢測(cè)儀可以精確地檢測(cè)蛇毒或其他毒素的溶膜活性,在工業(yè)化生產(chǎn)IgY方面有較好的應(yīng)用前景。 3、經(jīng)水稀釋法提取分離抗體,純度可達(dá)電泳純,抗體活性強(qiáng),回收率高、活性好,方法經(jīng)濟(jì)、簡(jiǎn)單易行。 4、口服制劑的抗蛇毒IgY,若能增加其免疫抗原的種類(lèi),抗體純化程度更高、效價(jià)更高,則其保護(hù)作用和應(yīng)用范圍將得以極大擴(kuò)展。
[Abstract]:Background: the anti snake venom antibody can be divided into two kinds, antivenene (AV) and anti venom chicken egg yolk antibody (immunoglobulins in yolk, IgY). The former is isolated from the blood of mammalian and the latter is extracted from the yolk of poultry. Since 1990s, foreign scholars have prepared the anti cobra, Ru from the chicken egg yolk. Ssell's viper, Saw-scaled viper, B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and corresponding venom are effective in the domestic, anti Cobra king snake, anti Agkistrodon snake, the preparation and characteristics of the anti Cobra and viper venom. Compared to the antibodies in the blood (such as rats, rabbits, pigs, horses and cattle), the yolk antibody has an incomparable superiority [9,10] (Schade et al.1992; Larsson et al., 1991): not combining with protein G or protein A, not activating the complement system, not combining with the rheumatoid factor, avoiding the false negative and false positive problems in the immunoassay; the egg source side The production cost is low and suitable for large-scale production; the egg yolk produced by the immune hen has only one antibody, with good homogenization, stable physicochemical properties, acid resistance, heat resistance, easy preservation and convenient transportation and use. In addition, the chicken is far from the mammalian species, and a strong immune response is easily produced when a small amount of spiny is stimulated. A kind of Southern China area is developed. A variety of venomous snakes are common snakes and prevention, treatment preparation, multivalent AV, can be used immediately in the case of venomous snakes without the need for snakebite, in order to win the best time for the best treatment, to avoid the occurrence of serious diseases and to reduce the mortality rate. In this laboratory, we have first prepared the monovalent anti Cobra and the viper IgY, which was first prepared in our laboratory. The aim of the experiment is to further develop the bivalent anti snake egg yolk antibody, so as to provide a new way for rescuing snakebite.
Objective: to prepare bivalent anti Cobra and viper chicken egg yolk antibody and to detect its biological activity, and to observe the protective effect of its oral preparation on Cobra and viper injured mice, and provide a new method for the prevention and treatment of snake wound, and lay a foundation for replacing horse derived serpent sera.
Method:
Preparation and extraction of bivalent anti snake venom IgY
1, the preparation of a bivalent anti snake venom IgY for preparation of the appropriate immunization scheme, using the patent - snake venom device to prepare the antigenic cobra, viper venom, fully emulsified the emulsifier self-made by 1:1 (V/V) and emulsified snake venom with FF's complete adjuvant in the first immunization, and then emulsified snake venom with FF's incomplete adjuvant. At the first third weeks after the first immunization, the immunization was carried out in 1 times at the fifth week, and the immunization dose was intermittent and gradually added. The two single antigens were alternately injected into the single chicken in order (double wing, chest, abdomen and back subcutaneous and muscle multipoint injection), and the interval of injection interval was one week.
2, bivalent anti snake venom IgY was detected by bi-directional immuno diffusion and immunoelectrophoresis to identify specific antibodies in the yolk; SDS-PAGE was used to determine the purity of the antibody products; the dynamic changes in the titer of specific IgY antibodies and the change in the titer of IgY in the plasma were detected by indirect ELISA.
3, the extraction of bivalent anti snake venom IgY was separated from egg white by yolk separator. The IgY antibody was extracted by water dilution.
Study on biological characteristics of two, bivalent anti snake venom IgY
1, the cross immunization of anti snake venom IgY and 10 common snake venom in China and its reaction intensity.2 were measured by bi-directional immuno diffusion method. The cobra and viper venom had different degree of membrane effect on the yolk membrane. The neutralization effect of bivalent anti snake venom IgY on the snake venom was observed by the homemade yolk membrane detector.
3, bivalent anti snake venom IgY was neutralized with cobra, viper venom in vitro, and intraperitoneal injection respectively. The neutralization effect of IgY on the lethal activity of snake venom was observed.
Three, bivalent anti venom IgY related animal experiments
1, bivalent anti snake venom IgY was administered to mice at high, medium and low concentrations at three concentrations. Blood separation plasma was taken at different time points, and the titer of bivalent anti snake venom IgY in the plasma of gavage mice was detected by indirect ELISA.
2, bivalent anti snake venom IgY was intragastric mice at high, middle and low three concentrations. The mice were intraperitoneally injected with cobra snakes and viper venom at different time points, and the protective effect of bivalent anti snake venom IgY on mice was detected.
Four, statistics were analyzed by t test and SPSS software.
Result:
1, snake venom has high activity and less impurity.
After the initial small dose of immunization, the specific yolk antibody can be detected in the yolk for ninth days. After the intermittent, the immunization is gradually added, the titer of the antibody continues to increase, and the high level can be maintained for a long time, and the effective price can be maintained for a long time.
2, the egg yolk separation collector (patent number: 2006200559807.3) was prepared during the experiment. Using this instrument, the egg yolk, egg white, the yolk separation degree was high, the yolk membrane was clean, the yolk membrane was clean, and the residual amount of egg white was less, which was beneficial to further separation and purification.
3, the water dilution method has good effect on degreasing. If further ammonium sulfate is salted out, the recovery rate of protein is about 40%., if the conditions can be further optimized, the antibody is purified by the method of sulfur eosinophilic chromatography, hydrophobic chromatography and gel chromatography, and the higher purity of antibody is expected.
4, by the bi-directional immuno diffusion method, the bivalent anti snake venom IgY has the most obvious reaction with the Zhoushan Cobra and the king cobra. The reaction precipitation line is the clearest, and the cross reaction is not equal to the venom of the cobra subfamily and viper subfamily, and the venom of the Agkistrodon hutpistrodon hkhaghistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackistrodon ackkistrodon ackkistrodon ackistrodon hkhaghagi Agkistrodon acutus.
5, according to the time results of the yolk membrane dissolved film detector, a certain amount of water extract can protect against snake venom IgY.
6, in vitro neutralization experiments showed that a certain concentration of water extracts against snake venom IgY could completely protect the experimental mice from 2 times the attack of LD50 Cobra and viper.
7, the anti snake venom IgY with different concentrations can have different protective effects on the cobra and viper injured mice. When the dose of IgY is high enough, it can completely protect the mice from the attack of the cobra and viper venom.
1, the Brunei hens were immunized with cobra and viper venom. After the first small dose, intermittent, and gradually increased immunity, the chicken yolk antibody with high effective price and specific antibody was obtained for several weeks.
2, the homemade yolk separation collector can separate the yolk in batch, and the yolk membrane detector can accurately detect the membrane activity of the snake venom or other toxin. It has a good application prospect in the industrial production of IgY.
3, the extraction and isolation of antibodies by water dilution method can achieve the purity of electrophoresis, strong antibody activity, high recovery rate and good activity. The method is economical and simple.
4, the protective effect and application range of the antivenom IgY of oral preparation will be greatly expanded if it can increase the type of its immune antigen, the degree of antibody purification is higher and the titer is higher.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

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