人源抗狂犬病毒基因工程抗體研究
發(fā)布時(shí)間：2019-05-16 19:12
【摘要】：狂犬病是由狂犬病毒(Rabies Virus, RV)引起的人獸共患疾病,發(fā)病后死亡率幾乎達(dá)百分之百。狂犬病毒屬?gòu)棤畈《究?Rhabdoviridae)狂犬病毒屬(Lyssavirus),基因組編碼五種結(jié)構(gòu)蛋白,其中由G基因編碼的糖蛋白,是病毒與宿主細(xì)胞結(jié)合的配體,能誘導(dǎo)機(jī)體產(chǎn)生中和抗體,與病毒的毒力、致病性密切相關(guān)。根據(jù)世界衛(wèi)生組織(World Health Organization, WHO)推薦,對(duì)狂犬病III級(jí)暴露后的預(yù)防主要是采用狂犬疫苗注射結(jié)合抗狂犬病毒免疫球蛋白(rabies immune globulin, RIG)的方法。目前使用的兩類(lèi)RIG為人抗狂犬病毒免疫球蛋白(human rabies immune globulin, HRIG)和馬抗狂犬病毒免疫球蛋白(Equine rabies immune globulin, ERIG)。但ERIG副反應(yīng)比較嚴(yán)重,而且對(duì)某些疫苗的抗體反應(yīng)有抑制,HRIG價(jià)格昂貴,供應(yīng)量有限并且有潛在的病原威脅。抗狂犬病毒單克隆抗體(monoclonal antibodies, mAbs)具有中和效果高,安全性好,成本低,可大量生產(chǎn)等優(yōu)點(diǎn),能夠取代RIG用于狂犬病的暴露后預(yù)防。本研究以噬菌體表面展示技術(shù)為平臺(tái),篩選人源抗狂犬病毒中和抗體,實(shí)驗(yàn)有以下三部分：一,人源抗狂犬病毒Fab噬菌體抗體庫(kù)的構(gòu)建與篩選我們首先采集具有高滴度狂犬病毒抗體的疫苗注射者外周血,分離淋巴細(xì)胞,然后提取總RNA并合成cDNA,接著用特異性的PCR引物擴(kuò)增特異性輕鏈和重鏈Fd段基因,在對(duì)PCR產(chǎn)物鑒定和純化后,用輕鏈基因與噬菌粒pComb3構(gòu)建了輕鏈庫(kù),隨后將重鏈基因克隆入輕鏈庫(kù)中構(gòu)建Fab噬菌體抗體庫(kù)。最后使用輔助噬菌體對(duì)Fab噬菌體抗體庫(kù)進(jìn)行包裝,檢測(cè)庫(kù)容量,結(jié)果表明我們所構(gòu)建的Fab噬菌體抗體庫(kù)的容量為4.5×108,輕鏈插入率和重鏈插入率均為100%,完全滿(mǎn)足我們篩庫(kù)的需要。在成功構(gòu)建Fab噬菌體抗體庫(kù)的基礎(chǔ)上,用純化的狂犬病毒顆粒aG和CTN株對(duì)Fab噬菌體抗體庫(kù)進(jìn)行富集篩選,利用ELISA、IFA對(duì)所獲人源單克隆抗體Fab段基因的功能特性進(jìn)行鑒定,并通過(guò)序列測(cè)定確定所獲抗體的基因序列,然后與Genebank報(bào)道的抗體序列進(jìn)行比較,根據(jù)抗體基因序列推斷出其氨基酸序列,與vbase database比較,確定其CDR區(qū)。結(jié)果我們獲得11株Fab抗體,序列測(cè)定結(jié)果證明其序列均為新的抗體序列,間接免疫熒光試驗(yàn)證明均針對(duì)狂犬病毒糖蛋白。二,人源抗狂犬病毒全抗體表達(dá)及功能鑒定在原核表達(dá)Fab抗體的基礎(chǔ)上,我們利用昆蟲(chóng)桿狀病毒載體技術(shù)平臺(tái)實(shí)現(xiàn)了全抗體的真核表達(dá)。我們將11株Fab抗體中5株Fab抗體(RVFab1、RVFab3、 RVFab5、RVFab8、RVFab9)的輕鏈和重鏈Fd段基因插入桿狀病毒載體pAC-L-Fc,然后將構(gòu)建好的重組桿狀病毒質(zhì)粒轉(zhuǎn)染昆蟲(chóng)細(xì)胞,通過(guò)IFA檢測(cè)轉(zhuǎn)染效果,接著純化和擴(kuò)增重組病毒,對(duì)表達(dá)產(chǎn)物進(jìn)行純化。利用SDS-PAGE、 ELISA、IFA等方法純化的抗狂犬病毒IgG全抗進(jìn)行功能鑒定,結(jié)果表明5株人源單克隆抗體均特異性針對(duì)狂犬病毒糖蛋白。利用非競(jìng)爭(zhēng)ELISA檢測(cè)5株抗體的親和力,均高達(dá)10-9M。利用快速免疫熒光灶抑制中和試驗(yàn)對(duì)單抗的中和活性進(jìn)行檢測(cè),結(jié)果表明5株單抗均具有較好的中和活性。三,人源抗狂犬病毒中和抗體表位研究通過(guò)競(jìng)爭(zhēng)ELISA對(duì)5株抗體與抗原結(jié)合表位的相互關(guān)系進(jìn)行鑒定,結(jié)果表明,5株抗體所識(shí)別的抗原表位彼此間存在相互重疊的關(guān)系。進(jìn)一步利用競(jìng)爭(zhēng)ELISA對(duì)5株抗體與一株針對(duì)狂犬病毒糖蛋白I號(hào)中和表位的抗體RVIgG57進(jìn)行鑒定,結(jié)果表明,5株抗體與RVIgG57所識(shí)別的抗原表位均不存在相互重疊的關(guān)系。通過(guò)生物信息學(xué)分析狂犬病毒糖蛋白關(guān)鍵抗原位點(diǎn),并結(jié)合相關(guān)文獻(xiàn)報(bào)道,設(shè)計(jì)針對(duì)狂犬病毒糖蛋白三個(gè)主要中和位點(diǎn)的點(diǎn)突變,通過(guò)IFA分析人源抗體與中和位點(diǎn)突變體的結(jié)合活性。結(jié)果表明：我們獲得的5株抗體均針對(duì)狂犬病毒糖蛋白II號(hào)中和表位,而RVIgG57針對(duì)狂犬病毒糖蛋白I號(hào)中和表位,與文獻(xiàn)描述一致。綜上所述,本研究運(yùn)用噬菌體抗體庫(kù)技術(shù),成功構(gòu)建了人源抗狂犬病毒Fab噬菌體抗體庫(kù),篩選獲得了11株特異性針對(duì)狂犬病毒糖蛋白的人源Fab抗體。將其中5株構(gòu)建為IgG全抗體,顯示均具有高親和力和高中和活性,并且針對(duì)狂犬病毒糖蛋白II號(hào)中和抗原表位。這一結(jié)果的獲得為單克隆抗體取代RIG做為狂犬病暴露后預(yù)防的被動(dòng)免疫制劑奠定了基礎(chǔ)。
[Abstract]:Rabies are caused by the rabies virus (RV), and the mortality rate is almost 100% after the onset of the disease. The rabies virus belongs to the genus Rhabdoviridae, and the genome encodes five structural proteins, wherein the glycoprotein encoded by the G gene is a ligand which is bound by the virus and the host cell, can induce the production of neutralizing antibodies in the body, and is closely related to the virulence and the pathogenicity of the virus. According to the recommendations of the World Health Organization (WHO), the prevention of rabies III exposure is mainly the use of rabies vaccine injection in combination with the method of anti-rabies virus immune globulin (RIG). Two types of RIG are currently used as human anti-rabies virus immune globulin (HRG) and horse anti-rabies virus immunoglobulin (ERIG). However, the side reactions of the ERIG are serious, and the antibody response to some vaccines is inhibited, and the HRG is expensive, the supply is limited, and the potential pathogenic threat is present. The anti-rabies virus monoclonal antibody (mAbs) has the advantages of high neutralization effect, good safety, low cost, large production and the like, and can replace the RIG for preventing the rabies. in that study, the phage surface display technology is used as a platform to screen the human anti-rabies virus neutralizing antibody, and the experiment has the following three parts:1. the construction and the screening of a human anti-rabies virus Fab phage antibody library first collect the peripheral blood of the vaccine injection with the high-titer rabies virus antibody, the lymphocyte is separated, the total RNA is extracted and the cDNA is synthesized, the specific light chain and the heavy chain Fd segment gene are amplified by specific PCR primers, and the light chain library is constructed by the light chain gene and the phagemid pComb3 after the PCR product is identified and purified; The heavy chain gene was then cloned into the light chain library to construct the Fab phage antibody library. Finally, using the auxiliary phage to package the Fab phage antibody library, the library capacity was detected. The results showed that the capacity of the Fab phage antibody library was 4.5-108, the light chain insertion rate and the heavy chain insertion rate were 100%, which completely met the needs of our screen library. on the basis of successfully constructing the Fab phage antibody library, the Fab phage antibody library is enriched and screened by purified rabies virus particles aG and CTN strains, and the functional characteristics of the obtained Fab fragment genes of the human monoclonal antibody are identified by ELISA and IFA, The gene sequence of the obtained antibody is determined by the sequence measurement, and then compared with the antibody sequence reported by Genbank, the amino acid sequence of the antibody is deduced according to the sequence of the antibody gene, and the CDR region thereof is determined based on the comparison with the vbase database. As a result,11 Fab antibodies were obtained, and the results of the sequence determination showed that the sequence was a new antibody sequence, and the indirect immunofluorescence test proved to be specific to the rabies virus glycoprotein. 2. The full-antibody expression and function of the human anti-rabies virus are based on the prokaryotic expression of the Fab antibody, and the eukaryotic expression of the whole antibody is realized by using the insect baculovirus vector technology platform. The light chain and the heavy chain Fd section gene of the five Fab antibodies (RVFab1, RVFab3, RVFab5, RVFab8, RVFab9) in the 11 Fab antibodies were inserted into the baculovirus vector pAC-L-Fc, then the constructed recombinant baculovirus plasmid was transfected into the insect cell, the transfection effect was detected by IFA, and then the recombinant virus was purified and amplified, The expression product was purified. The anti-rabies virus IgG was purified by SDS-PAGE, ELISA, IFA and other methods. The results showed that 5 strains of human monoclonal antibodies were specific to the glycoprotein of rabies virus. The affinity of five antibodies was detected by non-competitive ELISA, which was as high as 10-9M. The neutralization test was used to detect the neutralizing activity of the monoclonal antibody, and the results showed that the 5 McAbs had better neutralizing activity. 3. The anti-rabies virus and the antibody epitope of the human anti-rabies virus were identified by the competitive ELISA. The results showed that the antigen epitopes recognized by the five anti-rabies virus were in overlapping relationship with each other. The anti-rabies virus glycoprotein I and the antibody RVIgG57 were identified by competitive ELISA. The results showed that there were no overlapping relationship between the five antibody and the antigen epitope recognized by the RVIgG57. The key antigenic site of the rabies virus glycoprotein was analyzed by bioinformatics, and the point mutation of the three main neutralizing sites of the rabies virus glycoprotein was designed and the binding activity of the human antibody with the neutralizing site was analyzed by IFA. The results showed that 5 of the antibodies we obtained were for the neutralizing epitope of the rabies virus glycoprotein II, while the RVIgG57 is the neutralizing epitope of the rabies virus glycoprotein I and is consistent with the literature description. In conclusion, the phage antibody library was successfully constructed by using the phage antibody library technique, and 11 human Fab antibodies specific to the rabies virus glycoprotein were selected. Five of these were constructed as IgG full-antibodies, showing high affinity and high school and activity, and for rabies virus glycoprotein II and epitope. The result is that the obtained monoclonal antibody replaces the RIG as the basis for the passive immune preparation after the rabies exposure.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
本文編號(hào)：2478502
[Abstract]:Rabies are caused by the rabies virus (RV), and the mortality rate is almost 100% after the onset of the disease. The rabies virus belongs to the genus Rhabdoviridae, and the genome encodes five structural proteins, wherein the glycoprotein encoded by the G gene is a ligand which is bound by the virus and the host cell, can induce the production of neutralizing antibodies in the body, and is closely related to the virulence and the pathogenicity of the virus. According to the recommendations of the World Health Organization (WHO), the prevention of rabies III exposure is mainly the use of rabies vaccine injection in combination with the method of anti-rabies virus immune globulin (RIG). Two types of RIG are currently used as human anti-rabies virus immune globulin (HRG) and horse anti-rabies virus immunoglobulin (ERIG). However, the side reactions of the ERIG are serious, and the antibody response to some vaccines is inhibited, and the HRG is expensive, the supply is limited, and the potential pathogenic threat is present. The anti-rabies virus monoclonal antibody (mAbs) has the advantages of high neutralization effect, good safety, low cost, large production and the like, and can replace the RIG for preventing the rabies. in that study, the phage surface display technology is used as a platform to screen the human anti-rabies virus neutralizing antibody, and the experiment has the following three parts:1. the construction and the screening of a human anti-rabies virus Fab phage antibody library first collect the peripheral blood of the vaccine injection with the high-titer rabies virus antibody, the lymphocyte is separated, the total RNA is extracted and the cDNA is synthesized, the specific light chain and the heavy chain Fd segment gene are amplified by specific PCR primers, and the light chain library is constructed by the light chain gene and the phagemid pComb3 after the PCR product is identified and purified; The heavy chain gene was then cloned into the light chain library to construct the Fab phage antibody library. Finally, using the auxiliary phage to package the Fab phage antibody library, the library capacity was detected. The results showed that the capacity of the Fab phage antibody library was 4.5-108, the light chain insertion rate and the heavy chain insertion rate were 100%, which completely met the needs of our screen library. on the basis of successfully constructing the Fab phage antibody library, the Fab phage antibody library is enriched and screened by purified rabies virus particles aG and CTN strains, and the functional characteristics of the obtained Fab fragment genes of the human monoclonal antibody are identified by ELISA and IFA, The gene sequence of the obtained antibody is determined by the sequence measurement, and then compared with the antibody sequence reported by Genbank, the amino acid sequence of the antibody is deduced according to the sequence of the antibody gene, and the CDR region thereof is determined based on the comparison with the vbase database. As a result,11 Fab antibodies were obtained, and the results of the sequence determination showed that the sequence was a new antibody sequence, and the indirect immunofluorescence test proved to be specific to the rabies virus glycoprotein. 2. The full-antibody expression and function of the human anti-rabies virus are based on the prokaryotic expression of the Fab antibody, and the eukaryotic expression of the whole antibody is realized by using the insect baculovirus vector technology platform. The light chain and the heavy chain Fd section gene of the five Fab antibodies (RVFab1, RVFab3, RVFab5, RVFab8, RVFab9) in the 11 Fab antibodies were inserted into the baculovirus vector pAC-L-Fc, then the constructed recombinant baculovirus plasmid was transfected into the insect cell, the transfection effect was detected by IFA, and then the recombinant virus was purified and amplified, The expression product was purified. The anti-rabies virus IgG was purified by SDS-PAGE, ELISA, IFA and other methods. The results showed that 5 strains of human monoclonal antibodies were specific to the glycoprotein of rabies virus. The affinity of five antibodies was detected by non-competitive ELISA, which was as high as 10-9M. The neutralization test was used to detect the neutralizing activity of the monoclonal antibody, and the results showed that the 5 McAbs had better neutralizing activity. 3. The anti-rabies virus and the antibody epitope of the human anti-rabies virus were identified by the competitive ELISA. The results showed that the antigen epitopes recognized by the five anti-rabies virus were in overlapping relationship with each other. The anti-rabies virus glycoprotein I and the antibody RVIgG57 were identified by competitive ELISA. The results showed that there were no overlapping relationship between the five antibody and the antigen epitope recognized by the RVIgG57. The key antigenic site of the rabies virus glycoprotein was analyzed by bioinformatics, and the point mutation of the three main neutralizing sites of the rabies virus glycoprotein was designed and the binding activity of the human antibody with the neutralizing site was analyzed by IFA. The results showed that 5 of the antibodies we obtained were for the neutralizing epitope of the rabies virus glycoprotein II, while the RVIgG57 is the neutralizing epitope of the rabies virus glycoprotein I and is consistent with the literature description. In conclusion, the phage antibody library was successfully constructed by using the phage antibody library technique, and 11 human Fab antibodies specific to the rabies virus glycoprotein were selected. Five of these were constructed as IgG full-antibodies, showing high affinity and high school and activity, and for rabies virus glycoprotein II and epitope. The result is that the obtained monoclonal antibody replaces the RIG as the basis for the passive immune preparation after the rabies exposure.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
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