本文選題：登革病毒 + 單克隆抗體　； 參考：《第三軍醫(yī)大學》2008年博士論文

【摘要】： 登革病毒(Dengue Virus,DENV)是黃病毒屬、有包膜的單股正鏈RNA病毒,由于包膜蛋白的抗原性不同,又分為四種血清型,即DENV1～4。DENV主要以埃及伊蚊和白紋伊蚊為媒介而廣泛流行于熱帶和亞熱帶地區(qū)。每年,DENV會導致數(shù)百萬人感染,引起登革熱(dengue classical fever,DF)和登革出血熱/休克綜合癥(dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS)。DF是自限性發(fā)熱性疾病;而DHF/DSS則是威脅患者生命的重癥,其主要特征是血管通透性顯著增加,導致血漿滲漏。每年有大約50萬DHF/DSS患者需要及時入院治療,否則病死率高達50 %。然而,DHF/DSS的發(fā)病機制,以及DENV與宿主細胞之間相互作用的機制尚不清楚。 雖然在過去幾十年中,研究者從多方式、多角度入手研制DENV的疫苗,但至今仍沒有預防和治療DENV感染的疫苗和藥物。無論是減毒活疫苗、多價疫苗,還是核酸疫苗、蛋白成分疫苗都遇到DENV流行株抗原變異、抗體增強效應(antibody dependent enhancement,ADE)等障礙而進展緩慢。 基于近年來DHF/DSS患者數(shù)量明顯增加和上述研究現(xiàn)狀,被動免疫或者說是治療性單克隆抗體(Monoclonal antibodies,mAb)再次引起了研究者的關注。早在1907年人體抗血清就被用來治療麻疹病毒感染的患者。20世紀40年代起,從高免疫的血清中提取的免疫球蛋白來預防和治療病毒感染成為熱點,部分制品至今還被用于臨床治療。而特異性不高、成分復雜和潛在的疾病傳播途徑等缺陷極大的限制了此類生物制劑的使用。1982年mAb技術的出現(xiàn)后,研發(fā)高中和特性的mAb用于預防和治療病毒性疾病就受到研究者的青睞。特別是進入21世紀以來,生物技術和工業(yè)技術的進步,大量的治療性mAb藥物已進入臨床實驗或已應用于臨床,成為生物制藥中增長最快的部分。治療性mAb成為防治病毒的新手段,新焦點。 因此,無論從研究DENV病理機制角度,還是從研發(fā)治療性mAb角度,獲得多株特異性mAb具有重要意義。特別是具有高中和能力的mAb,是研究致病機制的免疫學工具和治療性mAb的基本材料。本試驗首先通過蔗糖密度梯度高速離心獲得純化的DENV2(Tr1751)株的具感染活性的病毒顆粒,并利用純化的病毒顆粒免疫SPF級BALB/c小鼠。之后通過雜交瘤細胞技術獲得分泌DENV特異性mAb的雜交瘤細胞株10株,78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1、8H4。然后分別對10株mAb進行性質鑒定,其中具有中和能力的mAb有7株(2B10、78#、1E7、7F7、8B6、8F12、8H4)包括2株中和能力較高的mAbs(78#、8F12),并對其在體內(nèi)外的保護活性進行驗證。結果提示通過全病毒免疫來制備中和抗體具有一定的優(yōu)勢,獲得的mAbs也為后續(xù)研究DENV的致病機理,以及治療性mAb的研發(fā)提供了有利的免疫學工具。 本研究主要結果與結論如下: 1.建立了純化的DENV2的方法,獲得具有感染性的純化病毒 本實驗通過DENV2的天然中介宿主細胞白紋伊蚊C6/36細胞增殖出大量病毒,并測定病毒滴度。除去細胞碎片后,使用PEG-8000對病毒進行濃縮,測定濃度病毒滴度。最后通過蔗糖密度梯度高速離心來獲得純化的DENV2具感染性的病毒顆粒,并通過病毒滴度測定和SDS-PAGE來驗證純化病毒及其含量。通過不同蔗糖密度梯度比較,確定了DENV2主要沉降區(qū)帶,建立了純化DENV的方法,并制備了一定量的純化病毒供下述實驗使用。 2.通過雜交瘤技術獲得10株鼠源性抗DENV2的mAbs 利用純化的DENV2病毒顆粒免疫SPF級的BALB/c小鼠10只,細胞融合前取小鼠尾血進行細胞爬片間接免疫熒光染色,檢測特異性抗體產(chǎn)生情況。無菌條件下取出免疫效果好的小鼠脾臟,機械分離為單個細胞后與小鼠骨髓瘤細胞株SP2/0在融合劑PEG-4000的作用下形成雜交瘤細胞。通過HAT培養(yǎng)篩選、間接ELISA陽性篩選和反復單克隆化等方法,獲得了10株分泌DENV2特異性mAbs的穩(wěn)定雜交瘤細胞株,命名為78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1、8H4。以無血清1640培養(yǎng)液分別培養(yǎng)各雜交瘤細胞來獲得濃度較低的mAb或通過小鼠腹腔注射培養(yǎng)來獲得大量較高濃度的mAb腹水,供下述實驗使用。 3. 10株mAbs性質的鑒定 (1)間接ELISA驗證10株mAbs的特異性。以純化的DENV2為抗原包被96孔板,同時用1 % BSA包被96孔板作為陰性對照。以mAb上清液或腹水為一抗,HRP-山羊抗小鼠IgG為二抗,OPD顯色。終止顯色后測定OD492nm。以吸光值大于對照值2.1倍為陽性。制備的10株mAbs上清或腹水反映強弱各不相同,但均顯示為陽性。 (2)通過間接ELISA進行抗體飽和曲線的繪制。實驗步驟同前,首先確定抗原最適包被濃度,然后將各株mAb腹水倍比稀釋后作為一抗,顯色后繪制OD492nm曲線,通過分析抗體飽和曲線得出各株抗體飽和稀釋度。對于識別相同病毒蛋白,并且抗體亞型相同的mAbs,我們通過抗體疊加實驗來驗證其識別的抗原位點是否重疊。將這些mAbs稀釋到各自的飽和濃度后,分別進行ELISA疊加實驗,結果顯示均出現(xiàn)較明顯的疊加陽性,證實它們識別不同的病毒抗原位點。 (3)間接免疫熒光實驗測定各mAb對天然病毒抗原的識別情況。將感染DENV2后發(fā)病的幼鼠腦制成組織切片進行間接免疫熒光測定,發(fā)現(xiàn)8株mAbs(78#、1E7、2B10、6C1、7F7、8B6、8F12、8H4)能出現(xiàn)特異性熒光,而2株mAbs(6H3、8H1)沒有特異性反應出現(xiàn)。 (4)Western-blot確定各株mAb識別的病毒蛋白。純化病毒經(jīng)過SDS-PAGE后,半干轉印到醋酸纖維膜上,按泳道剪條后,分別孵育各mAb,以HRP-山羊抗小鼠IgG為二抗,DAB顯色。10株mAbs中有6株(78#、1E7、2B10、8B6、8F12、8H1)識別DENV2 E蛋白,1株(6C1)識別非結構蛋白NS3,2株(8H1、6H3)識別非結構蛋白NS5,1株(7F7)無特異性反應條帶出現(xiàn)。 (5)其它指標:通過Sigma抗體分型試劑盒對各株mAb的抗體型和亞型進行鑒定。通過Giemsa染色確定雜交瘤細胞的染色體數(shù)量,檢測雜交瘤細胞傳代或復蘇后的細胞穩(wěn)定狀態(tài)。 4. PRNT測定10株mAbs的中和活性 以非洲綠猴Vero細胞為測試細胞,mAb-DENV2混合物為感染源,正常鼠血清(normal mouse sera,NMS)-DENV2混合物為陰性對照,測定50 %病毒中和濃度——PRNT50。 待Vero細胞在24孔板中單層覆蓋生長后,將mAb(或NMS)倍比稀釋1:10、1:20、1:40、1:80、1:160、1:320。加入等體積的病毒稀釋液后,mAb(或NMS)的實際稀釋度為1:20、1:40、1:80、1:160、1:320、1:640�？刂萍尤氲牟《玖�,1 ml混合物中含有約1000 PFU的DENV2。mAb-DENV2(或NMS- DENV2)混合物37°C水浴1 h后,每孔200μl加入各孔中37°C孵育1.5 h。每孔約含有200 PFU的病毒和不同稀釋度的抗體(或NMS)。之后以含1.2 %的甲基纖維素的MEM培養(yǎng)基培養(yǎng)7天,再通過結晶紫染色對病毒形成的噬斑(Plaque)進行計數(shù)。通過比對相同稀釋度NMS的陰性孔,觀察各株mAb有無中和作用,并計算中和抗體的PRNT50值。具有中和能力的mAb有7株:78#(1:60)、1E7(1:30)、2B10(1:35)、7F7(1:20)、8B6(1:30)、8F12(1:40)、8H4(1:15)。其中具有較高中和能力的2株mAbs:78#和8F12,兩者混合后的PRNT50達到1:80。 5. BALB/c乳鼠保護實驗 以BALB/c新生乳鼠(生后第2天)為DENV2挑戰(zhàn)及抗體體內(nèi)保護的研究對象,以體外實驗中具有較高中和能力的78#和8F12 mAbs為實驗對象,NMS為陰性對照。將mAb-DENV2或NMS-DENV2混合物37°C水浴1 h后,通過乳鼠腦內(nèi)注射,進行病毒挑戰(zhàn)實驗。在對高低兩個病毒進入量104 PFU和500 PFU進行比較的同時,分別對兩株mAbs的體內(nèi)保護性進行研究。發(fā)現(xiàn)它們均在一定程度上起到保護作用,乳鼠發(fā)病和死亡時間均比對照組延后。特別是在病毒進入量較低時,保護作用更為明顯。在上述實驗的基礎上,將兩株mAbs進行“cocktail”組合實驗。取500 PFU的病毒進入量,mAbs-DENV2于37°C水浴1 h后注射。結果顯示抗體的保護作用大幅度提高。乳鼠發(fā)病和死亡時間比對照組延后更顯著,并且有16.7 %的乳鼠被徹底保護(存活超過2周的觀察期)。 總之,本實驗建立了通過蔗糖密度梯度高速離心來純化DENV2的方法,并以純化病毒為抗原免疫BALB/c小鼠。通過免疫小鼠脾細胞與SP2/0細胞融合、篩選,獲得了10株DENV2特異性的mAbs。經(jīng)過性質鑒定,我們對10株mAbs所識別的病毒蛋白、抗體飽和度、免疫學活性等多方面進行性質鑒定。同時,通過PRNT對它們的中和特性進行檢測。在7株具有中和能力的mAbs中,78#和8F12具有較高的中和能力。進而又對這2株mAbs進行了BALB/c乳鼠保護實驗,結果提示它們具有一定的保護作用,而兩者的混合物保護作用更明顯。因此,10株mAbs是進一步研究DENV2感染機制的有效免疫學工具。同時,具有較好中和能力的mAbs也為后續(xù)研究治療性mAb的提供了初步的材料。
[Abstract]:Dengue Virus (DENV) is the genus of the yellow virus and the membrane of the single strand RNA virus. Because of the antigenicity of the envelope protein, it is divided into four serotypes. That is, DENV1 to 4.DENV is widely prevalent in the tropics and subtropics in the medium of Aedes aegypti and Aedes albopictus. Every year, DENV causes millions of people to infect and cause dengue. Dengue classical fever (DF) and dengue hemorrhagic fever / shock syndrome (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS) are self limiting febrile diseases; DHF/DSS is a serious threat to patients' life, the main feature of which is the significant increase in vascular permeability, resulting in plasma leakage. About 500 thousand of the patients are required each year. Hospitalization should be timely, or the mortality rate is as high as 50%. However, the pathogenesis of DHF/DSS and the mechanism of interaction between DENV and host cells are not yet clear.
Although in the past few decades, researchers have developed vaccines for DENV from multiple ways and multiple angles, but there are still no vaccines and drugs to prevent and treat DENV infection. Both live attenuated vaccines, polyvalent and nucleic acid vaccines, protein components vaccines meet the DENV epidemic strain variation, the antibody enhancement effect (antibody dependent enha) Ncement, ADE) and other obstacles have progressed slowly.
Based on the recent increase in the number of patients with DHF/DSS and the present situation, passive immunity or therapeutic monoclonal antibodies (Monoclonal antibodies, mAb) have attracted the attention of researchers again. People who were used to treat measles virus infection early in 1907 were raised from the high immune sera in the 40s of the.20 century. The immunoglobulin to prevent and treat viral infection has become a hot spot, and some of the products are still used for clinical treatment. But the specificity is not high, the complexity of the ingredients and the potential for the spread of the disease are greatly limited in the use of such biological agents in the use of.1982 mAb Technology, and the development of high school and characteristic mAb for prevention and treatment. Viral diseases are favored by researchers, especially since the twenty-first Century, the progress in biotechnology and industrial technology, a large number of therapeutic mAb drugs have entered clinical trials or have been applied to the clinic, becoming the fastest growing part of biopharmaceuticals. Therapeutic mAb has become a new and new focus for the prevention and treatment of viruses.
Therefore, it is of great significance to obtain multiple specific mAb from the study of DENV's pathological mechanism or from the perspective of developing therapeutic mAb. Especially, the mAb with high school and ability is an immunological tool to study the pathogenesis and the basic material for the therapeutic mAb. This experiment first passed the sucrose density gradient centrifugation to obtain the purified DENV. 2 (Tr1751) strain of the infected virus particles, and immunized SPF BALB/c mice with purified virus particles. After the hybridoma cell technique, 10 hybridoma cells secreting DENV specific mAb, 78#, 1E7,2B10,6C1,6H3,7F7,8B6,8F12,8H1,8H4., were then identified to 10 strains of mAb, which had neutralization ability. 7 strains of mAb (2B10,78#, 1E7,7F7,8B6,8F12,8H4) including 2 neutralized mAbs (78#, 8F12) and their protective activity in vivo and in vivo are verified. The results suggest that the neutralization antibody is prepared through the whole virus immunity. The obtained mAbs is also a continuation of the pathogenesis of DENV, as well as the development of therapeutic mAb. A beneficial immunological tool.
The main results and conclusions of this study are as follows:
1. a method of purifying DENV2 was established to obtain infectious purified virus.
In this experiment, a large number of viruses were proliferated by DENV2's natural medium host cells of Aedes albopictus C6/36 cells and the virus titer was measured. After removing the cell fragments, the virus was concentrated by PEG-8000 to determine the titer's titer. Finally, the purified DENV2 infected virus particles were obtained by sucrose density gradient high speed centrifugation. The virus titer and SDS-PAGE were used to verify the purified virus and its content. Through the comparison of different sucrose density gradient, the main subsidence zone of DENV2 was determined, the method of purifying DENV was established, and a certain amount of purified virus was prepared for the following experiment.
2. 10 strains of murine anti DENV2 mAbs were obtained by hybridoma technique.
The purified DENV2 virus particles were used to immunization 10 mice of SPF grade BALB/c mice. The mouse tail blood was taken to detect the specific antibody production by indirect immunofluorescence staining of the mouse tail blood before fusion. The spleen of mice with good immune effect was removed under aseptic conditions, and the machine was separated into a single cell and mouse myeloma cell line SP2/0 in the fusion agent P. Hybridoma cells were formed under the action of EG-4000. Through HAT culture screening, indirect ELISA positive screening and repeated monoclonal, 10 stable hybridoma cell lines secreting DENV2 specific mAbs were obtained, named 78#, and 1E7,2B10,6C1,6H3,7F7,8B6,8F12,8H1,8H4. was cultured in serum-free 1640 culture solution to obtain the hybridoma cells respectively. A high concentration of mAb ascites was obtained by low concentration of mAb or by intraperitoneal injection in mice for use in the following experiments.
Identification of the properties of 3.10 strains of mAbs
(1) the specificity of 10 strains of mAbs was verified by indirect ELISA. The purified DENV2 was coated with 96 orifice plates, and 1% BSA was used as a negative control with 96 orifice plates. MAb supernatant or ascites was a resistance, HRP- Goat anti mouse IgG was two, OPD coloured. The determination of OD492nm. was positive with 2.1 times more than 2.1 times the control value. The 10 mAbs prepared. Supernatants or ascites reflect strong or weak, but all show positive.
(2) draw the antibody saturation curve through indirect ELISA. The experiment step is first to determine the optimum concentration of the antigen, then the mAb ascites of each strain is diluted as one anti, and then the OD492nm curve is drawn after coloring, and the antibody saturation curve is analyzed. With the same subtype of mAbs, we tested the overlap of the antigens identified by the antibody superposition experiment. After diluting these mAbs to their respective saturated concentrations, the ELISA superposition experiment was carried out respectively. The results showed that there were obvious superposition positive, which confirmed the identification of different viral antigen loci.
(3) indirect immunofluorescence test was used to determine the identification of mAb for natural virus antigen. Indirect immunofluorescence was used to make tissue slices of young mice infected with DENV2 after infection. It was found that 8 strains of mAbs (78#, 1E7,2B10,6C1,7F7,8B6,8F12,8H4) could have specific fluorescence, and 2 strains of mAbs (6H3,8H1) had no specific reaction.
(4) Western-blot determined the virus protein identified by mAb. After the purification of the virus, the virus was transferred to the cellulose acetate membrane by SDS-PAGE, and after the strip was cut, the mAb was incubated respectively. The HRP- Goat anti mouse IgG was two, and 6 strains of DAB color.10 strain (78#, 1E7,2B10,8B6,8F12,8H1) identified the DENV2 protein, and 1 strains identified the non structural protein. 2 strains (8H1,6H3) identified non structural protein NS5,1 strain (7F7) with no specific reaction bands.
(5) other indicators: the Sigma antibody typing kit was used to identify the type and subtype of each strain of mAb. The number of chromosomes of hybridoma cells was determined by Giemsa staining, and the cell stability of hybridoma cells was detected and after resuscitation.
Determination of neutralization activity of 10 strains of mAbs by 4. PRNT
The mAb-DENV2 mixture was the source of infection, and the normal rat serum (normal mouse sera, NMS) -DENV2 mixture was negative control, and the neutralization concentration of 50% virus - PRNT50. was determined - PRNT50., taking the Vero cell of African green monkey as the test cell.
After the growth of a single layer of Vero cells in a 24 orifice plate, after adding mAb (or NMS) times the dilution 1:10,1:20,1:40,1:80,1:160,1:320. to the equal volume of viral diluents, the actual dilution of mAb (or NMS) is the amount of virus added by 1:20,1:40,1:80,1:160,1:320,1:640. control, and the 1 ml mixture contains about 1000 PFU DENV2.mAb-DENV2 (or NMS- DENV2). After the mixture of 37 C water bath 1 h, 200 mu l per pore was incubated at 37 degrees in each hole and 1.5 h. containing 200 PFU virus and different dilution antibodies (or NMS). Then, the MEM culture medium containing 1.2% methyl cellulose was cultured for 7 days, and then the virus plaque (Plaque) was counted by crystal violet staining. By comparison of the same dilution NMS. The negative pores were used to observe whether mAb had neutralization and calculated the PRNT50 value of neutralization antibody. The neutralizing mAb had 7 strains: 78# (1:60), 1E7 (1:30), 2B10 (1:35), 7F7, 8F12, 8F12, 8H4, of which 2 plants with higher neutralization capacity were mixed.
5. BALB/c milk rat protection experiment
The BALB/c neonatal rats (second days after birth) were the subjects of DENV2 challenge and antibody in vivo protection. The 78# and 8F12 mAbs with high neutralization ability in the experiment were tested and NMS was negative control. After the mixture of mAb-DENV2 or NMS-DENV2 mixture 37 degree C water bath 1 h, the test of virus challenge was carried out by intramuscular injection of the rat brain. The high and low two were high and low. In comparison with 104 PFU and 500 PFU, two strains of mAbs were studied in vivo. They were found to play a protective role to some extent, and the onset and death time of the mice were all longer than those of the control group. Two strains of mAbs were carried out in the "cocktail" combination test. The virus entry of 500 PFU was taken and mAbs-DENV2 was injected after 1 h in 37 C water bath. The results showed that the protective effect of the antibody was greatly improved. The onset and death time of the mice were more significant than that of the control group, and 16.7% of the mice were thoroughly protected (surviving for more than 2 weeks of observation).
In this experiment, the method of purifying DENV2 by sucrose density gradient high speed centrifugation was established and BALB/c mice were immunized with the purified virus as antigen. Through the fusion of mouse spleen cells and SP2/0 cells, 10 DENV2 specific mAbs. were identified. The virus proteins identified by 10 strains of mAbs and the saturation of antibody were obtained. They were identified in many aspects of immunological activity. Meanwhile, their neutralization properties were detected by PRNT. In 7 mAbs with neutralization capacity, 78# and 8F12 had higher neutralization ability. Then, the 2 mAbs was protected by BALB/c milk mice. The results suggest that they have a certain protective effect and a mixture of both. The protective effect is more obvious. Therefore, 10 mAbs strains are an effective immunological tool to further study the mechanism of DENV2 infection. At the same time, the better neutralizing mAbs also provides preliminary materials for the follow-up study of therapeutic mAb.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392

【共引文獻】

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