【摘要】： 狂犬病是由狂犬病病毒引起的人畜共患的烈性傳染病,也是迄今為止人類病死率最高的急性傳染病�？箍袢〔《締慰寺】贵w是研究狂犬病病毒診斷方法、治療及預(yù)防狂犬病的有力工具。為了獲得高效價(jià)、高特異性的抗狂犬病病毒的單克隆抗體,進(jìn)而將其應(yīng)用于開發(fā)一種方便、簡單、廉價(jià)、技術(shù)要求低且適合基層使用的診斷方法,本研究用人用狂犬疫苗免疫BALB/c小鼠,采用雜交瘤技術(shù)制備得到針對狂犬病病毒的穩(wěn)定的、效價(jià)高的、特異性強(qiáng)的單克隆抗體(mAb),并初步鑒定了雜交瘤和mAb的生物學(xué)特性,包括：雜交瘤的染色體、雜交瘤分泌抗體的穩(wěn)定性、mAb的效價(jià)、亞類、純度、蛋白濃度以及mAb的特異性。為開發(fā)簡單實(shí)用的檢測方法打下基礎(chǔ)。 一、融合條件的摸索 在充分了解SP2/0細(xì)胞系的生長、增殖和遺傳特性后,以聚乙二醇(PEG)誘導(dǎo)動物細(xì)胞融合,探索不同分子量、不同濃度的PEG作為融合劑對誘導(dǎo)SP2/0細(xì)胞與脾細(xì)胞融合的最適條件。結(jié)果表明分子量為4000,濃度為50%的PEG誘導(dǎo)的細(xì)胞融合率最高,為0.06429%,為下一步制備單克隆抗體奠定基礎(chǔ)。 二、單克隆抗體雜交瘤細(xì)胞株的制備、篩選、克隆、穩(wěn)定性及染色體 1.抗體制備與細(xì)胞融合 使用人用狂犬疫苗免疫雌性BALB/c小鼠,間隔2周免疫1次。雙方陣法確定了間接ELISA法的最佳抗原包被濃度為1：80,陽性血清最適工作稀釋度為1：400。間接ELISA法檢測抗體效價(jià)達(dá)到104的3d后,通過PEG4000使免疫B淋巴細(xì)胞和小鼠骨髓瘤細(xì)胞SP2/0融合。融合3-5d后出現(xiàn)雜交瘤細(xì)胞,7-10d后觀察到雜交瘤細(xì)胞克隆生長。經(jīng)HAT培養(yǎng)基選擇培養(yǎng)后,細(xì)胞的融合率為82.99%。 2.陽性克隆的篩選 融合后14d左右,融合的雜交瘤細(xì)胞長到大約培養(yǎng)板的1/2面積時(shí),按照已建立的間接ELISA法進(jìn)行篩選,陽性克隆率為15.90%(38/239),選擇OD值比較高的14株陽性雜交瘤細(xì)胞株進(jìn)行亞克隆。 3.陽性雜交瘤細(xì)胞的克隆化培養(yǎng) 用顯微操作法對陽性克隆的細(xì)胞株,分別進(jìn)行了亞克隆,得到17珠分泌抗狂犬病病毒單克隆抗體的雜交瘤細(xì)胞株,分別命名8F3、8F4、8G8、17A4、17A5、17A7、17B2、17B5、17B4、17C2、17C6、17D2、17D4、17D8、17E3、17E4、17F3。選擇OD450值較高,生長狀態(tài)良好的8F3、8F4、8G8、17B2、17E3、17E4、17F3進(jìn)行擴(kuò)大培養(yǎng)和鑒定,其它株冷凍保存。 4.抗體分泌的穩(wěn)定性檢測 將雜交瘤細(xì)胞連續(xù)培養(yǎng)傳代,共傳18代每隔3代檢測一次細(xì)胞培養(yǎng)上清的抗體效價(jià),結(jié)果顯示6代以后雜交瘤進(jìn)入穩(wěn)定分泌抗體時(shí)期。 5.雜交瘤細(xì)胞的染色體計(jì)數(shù) 雜交瘤細(xì)胞染色體的平均數(shù)為94條,符合融合細(xì)胞染色體的特點(diǎn)。 三、單克隆抗體的制備及初步鑒定 1.mAb的制備 通過收集培養(yǎng)上清、體內(nèi)誘生腹水、制備實(shí)體瘤分離血清法獲得并保存了8F3、8F4、8G8、17B2、17E3、17E4和17F3分泌的抗體。 2.mAb的純化 經(jīng)抗體亞類初步鑒定8F3、8F4、8G8、17B2、17E3、17E4、17F3所分泌的抗體類型皆屬于IgG亞類,采用辛酸-硫酸銨法進(jìn)行純化。 3.mAb的效價(jià) 間接ELISA法檢測培養(yǎng)上清、純化和未純化的腹水和實(shí)體瘤血清抗體效價(jià)。腹水和實(shí)體瘤血清中的抗體效價(jià)均高于104,最高的達(dá)1：128000,且腹水和實(shí)體瘤血清的效價(jià)比同種單抗細(xì)胞培養(yǎng)上清的效價(jià)均高100倍以上。 4.mAb的亞類 Ig類及亞類的鑒定結(jié)果顯示8F3和8F4屬于IgG2b,8G8和17E4屬于IgG3、17B2屬于IgG1,17E3和17F3屬于IgG2a。 5.mAb的純度 純化后的單抗在50kD和25kD附近出現(xiàn)兩條帶,與預(yù)期的IgG抗體重鏈和輕鏈條帶大小相符,而未純化的單抗泳道上則出現(xiàn)多條雜帶。 6.mAb的蛋白濃度 紫外分光光度法測得17B2、17E3、17E4、17F3、8F3、8F4、8G8、濃度分別為：1.6565mg/mL、0.0162mg/mL、0.0163mg/mL、1.7120mg/mL、1.6315mg/mL、1.6834mg/mL、0.0163mg/mL。 7.mAb的特異性 7株單克隆抗體與三個(gè)廠家的狂犬疫苗反應(yīng)均為陽性,與犬瘟熱病毒、犬細(xì)小病毒、犬傳染性肝炎病毒、犬副流感病毒、乙型肝炎病毒反應(yīng)均為陰性。說明7株單克隆抗體的特異性強(qiáng)且與其他病毒無交叉反應(yīng)。
[Abstract]:Rabies is a zoonotic infectious disease caused by rabies virus. It is also an acute infectious disease with the highest mortality of human beings. Monoclonal antibody against rabies virus is a powerful tool for the study of the diagnosis, treatment and prevention of rabies virus. In order to obtain high-cost, high-specificity anti-rabies virus monoclonal antibody. In this study, human rabies vaccine was used to immunize BALB/c mice, and hybridoma technology was used to prepare stable, potent and specific monoclonal antibodies (mAb) against rabies virus, and preliminary identification was carried out. The biological characteristics of hybridoma and mAb, including chromosome of hybridoma, stability of antibody secreted by hybridoma, titer of mAb, subclass, purity, protein concentration and specificity of mAb, were studied.
First, explore the conditions of convergence.
After fully understanding the growth, proliferation and genetic characteristics of SP2/0 cell line, PEG was used to induce the fusion of SP2/0 cells and spleen cells. The optimum conditions for inducing the fusion of SP2/0 cells and spleen cells with different molecular weights and concentrations of PEG were explored. 0.06429%, lay the foundation for further preparation of monoclonal antibodies.
Two, monoclonal antibody hybridoma cell line preparation, screening, cloning, stability and chromosomes.
1. antibody preparation and cell fusion
Female BALB/c mice were immunized with rabies vaccine at intervals of 2 weeks. The optimal antigen coating concentration of indirect ELISA was 1:80 and the optimum working dilution of positive serum was 1:400. After the titer of antibody reached 104 by indirect ELISA, SP2/C of B lymphocytes and mouse myeloma cells were immunized with PEG4000 3 days later. Hybridoma cells appeared 3-5 days after fusion and clonal growth of hybridoma cells was observed 7-10 days after fusion.
2. screening of positive clones
About 14 days after fusion, when the fused hybridoma cells grew to about 1/2 of the culture plate area, the positive cloning rate was 15.90% (38/239) by indirect ELISA method, and 14 hybridoma cell lines with high OD value were selected for subcloning.
3. cloning culture of positive hybridoma cells
Hybridoma cell lines secreting anti-rabies virus monoclonal antibodies from 17 beads were obtained by subcloning the positive clones with micromanipulation. The hybridoma cell lines were named 8F3, 8F4, 8G8, 17A4, 17A5, 17A7, 17B2, 17B5, 17B4, 17C2, 17C6, 17D2, 17D4, 17D8, 17E3, 17E4, 17F3. The selected OD450 values were high and the growth status was good. 3,17E4,17F3 was expanded and identified, and other plants were cryopreserved.
4. stability of antibody secretion
The antibody titers of the supernatant of hybridoma cells were detected every three generations in 18 passages. The results showed that the hybridoma entered a stable antibody secretion stage after 6 passages.
Chromosome count of hybridoma cells 5.
The average number of chromosomes of hybridoma cells is 94, which is consistent with the characteristics of the chromosomes of fusion cells.
Three, preparation and preliminary identification of monoclonal antibodies.
Preparation of 1.mAb
The antibodies secreted by 8F3, 8F4, 8G8, 17B2, 17E3, 17E4 and 17F3 were obtained and preserved by collecting culture supernatants and inducing ascites in vivo.
Purification of 2.mAb
The antibodies secreted by 8F3, 8F4, 8G8, 17B2, 17E3, 17E4 and 17F3 were identified as IgG subclasses and purified by octanoic acid-ammonium sulfate method.
Titer of 3.mAb
The titers of antibodies in ascites and solid tumor sera were higher than 104 and the highest was 1:128000. The titers of ascites and solid tumor sera were 100 times higher than those of monoclonal antibodies.
Subclasses of 4.mAb
The identification results of Ig class and subclass showed that 8F3 and 8F4 belonged to IgG2b, 8G8 and 17E4 belonged to IgG3, 17B2 belonged to IgG1, 17E3 belonged to IgG2a.
Purity of 5.mAb
The purified monoclonal antibody showed two bands near 50 kD and 25 kD, which were consistent with the expected IgG antibody heavy and light chain bands, while the unpurified monoclonal antibody showed multiple bands on the swimming track.
Protein concentration of 6.mAb
The concentrations of 17B2, 17E3, 17E4, 17F3, 8F3, 8F4 and 8G8 were 1.656565mg/mL, 0.0162mg/mL, 0.0163mg/mL, 1.7120mg/mL, 1.6315mg/mL, 1.6834mg/mL, 0.0163mg/mL, respectively.
Specificity of 7.mAb
All the 7 monoclonal antibodies reacted positively with rabies vaccines from three manufacturers, and were negative with canine distemper virus, canine parvovirus, canine infectious hepatitis virus, canine parainfluenza virus and hepatitis B virus.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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