【摘要】： 戊型肝炎(Hepatitis E, HE)是一種經(jīng)糞口途徑傳播的急性傳染病,在世界各地尤其是發(fā)展中國(guó)家廣泛流行,在發(fā)達(dá)國(guó)家也有散發(fā)。HE病死率約為0.4%,遠(yuǎn)較其他各型肝炎高,患者多為青壯年,孕期婦女病死率高達(dá)20%[1-2],給社會(huì)帶來(lái)了很大危害。近年來(lái)發(fā)現(xiàn)在豬等動(dòng)物中也有廣泛的戊型肝炎病毒(Hepatitis E Virus,HEV)感染,使該病成為一種人畜共患病[18]。 目前HE尚無(wú)特異的治療方法,也無(wú)主動(dòng)和被動(dòng)免疫制劑可供預(yù)防。國(guó)內(nèi)外學(xué)者均在致力于HEV疫苗的研究。而衡量疫苗是否有預(yù)防疾病作用的標(biāo)準(zhǔn),就是看疫苗能否誘導(dǎo)機(jī)體產(chǎn)生中和抗體。中和抗體不同于一般的抗體,它能與病毒結(jié)構(gòu)蛋白上的中和抗原表位結(jié)合,阻斷病毒與敏感細(xì)胞的結(jié)合,從而阻止該病毒入侵敏感細(xì)胞,使病毒失去感染性,對(duì)機(jī)體有保護(hù)作用,對(duì)疾病的診斷、預(yù)防和預(yù)后起著重要作用。檢測(cè)中和抗體的常用方法有動(dòng)物體內(nèi)試驗(yàn)和細(xì)胞培養(yǎng)體外試驗(yàn)兩種,但目前對(duì)HEV敏感的動(dòng)物只有非人靈長(zhǎng)類(lèi)動(dòng)物[6],使用非人靈長(zhǎng)類(lèi)動(dòng)物建立動(dòng)物模型,價(jià)格昂貴,方法繁瑣,試驗(yàn)周期長(zhǎng),動(dòng)物飼養(yǎng)要求高,耗費(fèi)大量的人力物力,難以在基層廣泛使用。另外,由于HEV缺乏有效的細(xì)胞培養(yǎng)體系,傳統(tǒng)的體外中和試驗(yàn)鑒定難以進(jìn)行,1997年Meng [10]等發(fā)明了基于PCR和細(xì)胞培養(yǎng)的體外中和試驗(yàn)并用于HEV中和抗體的檢測(cè),但該體外中和試驗(yàn)需要細(xì)胞培養(yǎng)及RT-PCR技術(shù),方法繁瑣,也難以普遍推廣應(yīng)用。針對(duì)HEV中和抗體檢測(cè)存在的問(wèn)題,本研究的目的是建立一種簡(jiǎn)便易行的檢測(cè)HEV中和抗體的方法,并進(jìn)行初步評(píng)價(jià)。本研究利用我們先前獲得的能夠穩(wěn)定分泌HEV中和性單克隆抗體(McAb)的雜交瘤細(xì)胞株,大量制備和純化HEV中和性McAb,并進(jìn)行酶標(biāo)記,使待測(cè)樣本中的HEV中和抗體與酶標(biāo)記HEV中和性McAb競(jìng)爭(zhēng)結(jié)合包被抗原上的HEV中和抗原表位,最后根據(jù)酶作用底物顯色反應(yīng)減弱的程度,實(shí)現(xiàn)對(duì)生物學(xué)樣本中的HEV中和抗體的競(jìng)爭(zhēng)酶聯(lián)免疫檢測(cè)。 本研究分為三個(gè)部分,具體研究結(jié)果如下: 一.HEV中和性單克隆抗體的大量制備、鑒定和純化本實(shí)驗(yàn)室通過(guò)B淋巴細(xì)胞雜交瘤技術(shù),以p166Chn和p166Bur免疫小鼠后獲得3株雜交瘤細(xì)胞株5G5、1G10和3G1。這3株細(xì)胞所分泌的McAb均不與單獨(dú)包被的GST發(fā)生結(jié)合,是針對(duì)HEV的特異性抗體。它們分泌的McAb能和7種不同基因型來(lái)源的p166均發(fā)生陽(yáng)性反應(yīng),為p166共同型McAb。用這3株雜交瘤細(xì)胞制備出大量的McAb,并對(duì)McAb進(jìn)行了HEV中和活性的鑒定,證實(shí)它們具有中和HEV的作用,是HEV中和性McAb。分別用正辛酸法,DEAE 52陰離子交換層析法,Protein-G親和層析法3種不同的方法對(duì)McAb進(jìn)行純化,并對(duì)這3種純化方法的結(jié)果進(jìn)行比較。我們將純化得到的產(chǎn)物系列稀釋后進(jìn)行間接ELISA檢測(cè)抗體滴度,顯示出用正辛酸法和Protein-G親和層析的方法純化的McAb滴度比DEAE 52陰離子交換層析法高,分別可達(dá)1:106(5G5),1:105(1G10)1:105(3G1)。經(jīng)SDS-PAGE,純化后的McAb僅在重、輕鏈位置出現(xiàn)明顯條帶,表明純化后的McAb純度較高。最終選用較經(jīng)濟(jì)、
[Abstract]:Hepatitis E (HE) is an acute infectious disease that is spread by the way of manure, and is widely popular in all parts of the world, especially in developing countries, and has also been distributed in developed countries. The mortality of HE is about 0.4%, which is much higher than that of other types of hepatitis. The mortality of pregnant women is 20%[1-2], which brings great harm to society. In recent years, it has been found that a wide range of hepatitis E virus (HEV) infection in pigs and other animals has caused the disease to become a human-animal disease[18]. At present, HE has no specific treatment method, nor is it active and is The dynamic immune preparation can be used for prevention, and both domestic and foreign scholars are committed to The study of the HEV vaccine, which measures whether the vaccine has a standard to prevent disease, is to see if the vaccine can be induced The neutralizing antibody is different from the general antibody, and can be combined with the neutralizing antigen epitope in the viral structural protein to block the binding of the virus and the sensitive cell, thereby preventing the virus from invading the sensitive cells, and protecting the body the effect, the diagnosis and prevention of the disease, and an animal model is established by using the non-human primate animal, the price is high, the cost is high, the method is complicated, the test period is long, the animal feeding requirement is high, a large amount of manpower and material resources are consumed, It is difficult to use extensively at the grass-roots level. In addition, due to the lack of an effective cell culture system in the HEV, the traditional in vitro neutralization test identification is difficult to carry out, and in 1997, Meng[10] et al. developed in vitro neutralization test based on the PCR and cell culture and used in the detection of the neutralizing antibody in the HEV, but the in vitro neutralization test requires cell culture and the method is complex, It is also difficult to popularize the application in general. The purpose of this study is to establish a simple and convenient method for detecting the neutralizing antibody in the HEV, aiming at the problems existing in the detection of the antibody and the antibody in the HEV. The present study makes use of the previously obtained hybridoma cell lines capable of stably secreting the neutralizing monoclonal antibody (McAb) of the HEV, a large number of preparation and purification of the HEV, and the sexual Mc and carrying out enzyme labeling so that the HEV in the sample to be tested and the antibody in the sample to be tested compete with the antibody and the sex McAb in the HEV to bind to the HEV in the antigen and the epitope of the antigen, and finally, the antibody and the antibody in the biological sample are realized according to the degree of the color reaction of the enzyme action substrate to decrease, competitive enzyme-linked immunosorbent assay. the present invention The study was divided into three parts, and the specific results were as follows: A. A large number of preparation, identification and purification of the neutralizing monoclonal antibody in the HEV were carried out by B-lymphocyte hybridoma technique, p166Chn and p166B. Three hybridoma cell lines 5G5, 1G10, and 3G1 were obtained after the mice were immunized. b is not bound to a separately coated GST, is a specific antibody against an HEV. The McAb secreted by them can be combined with 7 different groups A lot of McAb was prepared by using the three hybridoma cells, and the activity of the McAbs in the HEV was investigated. They were identified by positive caprylic acid method, DEAE 52 anion exchange chromatography and Protein-G affinity chromatography. The results of the three purification methods were compared, and the results of the three purification methods were compared. After the product family was diluted, the titer of the antibody was detected by indirect ELISA, and the concentration of the McAb of the purified McAb was higher than that of the DEAE 52 anion exchange chromatography with the method of n-octanoic acid and Protein-G affinity chromatography, respectively. 06 (5G5), 1: 105 (1G10) 1: 105 (3G1). in heavy and light chain position only
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R392

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