本文選題：高致病性禽流感病毒　切入點(diǎn)：治療性抗體　出處：《廈門(mén)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 高致病性禽流感(Avian influenza,AI)是由A型流感病毒H5N1引起的烈性傳染病。近年來(lái)隨著疾病的不斷蔓延,禽流感病毒不僅使各地養(yǎng)禽業(yè)蒙受巨大損失,而且還不斷突破種間屏障引發(fā)人感染事件。目前高致病性禽流感面臨的問(wèn)題是病死率居高不下且缺乏有效的治療藥物。由于流感病毒的持續(xù)快速變異,自然界中H5N1病毒已逐漸形成多種抗原性不同的變異亞系病毒株共流行的狀況,要滿足長(zhǎng)期戰(zhàn)略儲(chǔ)備藥物的需求,研制的H5N1治療藥物必須具備廣譜性。 中和單抗是防治病毒性疾病的一種有效手段,但由于禽流感病毒基因突變頻率高,特別是血凝素(HA)基因,因此針對(duì)禽流感病毒的中和單抗中和譜較窄,往往只能中和少數(shù)遺傳變異亞系的病毒,極大限制了這些中和單抗的應(yīng)用。 基于本實(shí)驗(yàn)室近期構(gòu)建的一個(gè)具有血凝抑制(AI)活性的H5特異性單克隆抗體庫(kù)(已經(jīng)用41株包含全部10個(gè)遺傳進(jìn)化分支的H5N1病毒代表株對(duì)這些單抗的HI活性和以小鼠為動(dòng)物模型的中和活性進(jìn)行了測(cè)定),本研究選取四株廣譜中和活性強(qiáng)的鼠單抗:4D1、10F7、8H5、13D4進(jìn)行人源化改造,以降低鼠抗在人治療過(guò)程中的HAMA反應(yīng),為這些抗體盡快進(jìn)入臨床應(yīng)用奠定基礎(chǔ);同時(shí)也希望建立一套治療性抗體人源化研究的平臺(tái)體系,包括基因的改造、表達(dá)載體的構(gòu)建、哺乳動(dòng)物細(xì)胞高效穩(wěn)定表達(dá)、無(wú)血清大規(guī)模培養(yǎng)、抗體純化工藝等。 本研究先以RT-PCR的方式從鼠雜交瘤細(xì)胞內(nèi)獲得四株抗體的可變區(qū)基因,再結(jié)合至人抗體IgG1的恒定區(qū),插入pcDNA3.1表達(dá)載體瞬時(shí)表達(dá)得到嵌合抗體產(chǎn)物;通過(guò)HI、ELISA、免疫熒光方法驗(yàn)證抗體具有活性。為了在短期內(nèi)累積一定的產(chǎn)物用于后續(xù)研究,我們選用FLP-IN定點(diǎn)整合表達(dá)系統(tǒng),其FRT定點(diǎn)整合的特點(diǎn)使抗體能夠穩(wěn)定高效表達(dá),經(jīng)過(guò)細(xì)胞篩選流程,我們現(xiàn)已獲得三株穩(wěn)定表達(dá)細(xì)胞株,而且抗體的表達(dá)產(chǎn)量提高了3-4倍。在獲得少量無(wú)血清培養(yǎng)的抗體純化物后,我們用多株H5N1病毒株HI實(shí)驗(yàn)、及細(xì)胞體外中和實(shí)驗(yàn)驗(yàn)證嵌合抗體的廣譜中和活性,以確定整個(gè)改造及表達(dá)過(guò)程中抗體的可變區(qū)活性沒(méi)有受到影響。 本研究選擇13D4嵌合抗體進(jìn)行大規(guī)模表達(dá)和純化。為了滿足下游抗體純化簡(jiǎn)便及大規(guī)模生產(chǎn)的需求,我們對(duì)CHO細(xì)胞的無(wú)血清培養(yǎng)方法也進(jìn)行了探索,包括優(yōu)化無(wú)血清培養(yǎng)基的營(yíng)養(yǎng)成分、滾瓶較大規(guī)模培養(yǎng)操作等。我們發(fā)現(xiàn)BSA添加物確實(shí)可以顯著提高嵌合抗體的產(chǎn)量,尤其是3%的添加比例,可使抗體產(chǎn)量提高8倍,但與此同時(shí)給抗體的純化帶來(lái)干擾,應(yīng)該綜合考慮抗體的純度和得率來(lái)決定最優(yōu)方案;蛋白A親和層析法一直是抗體純化的特異有效方式,我們摸索了不同洗脫P(yáng)H緩沖液,得到適合嵌合抗體純化的洗脫P(yáng)H值,最終純度達(dá)到95%,得率在90%。 針對(duì)具有代表性的四株H5N1病毒的動(dòng)物保護(hù)實(shí)驗(yàn)結(jié)果證明,在Vietnam/1194/2005感染后的第1和3天,以及在BH Goose/Qinghai/15c/2005和Shenzhen/406H/2006感染后的第1天,13D4嵌合抗體均有100%的治療效果。而在Indonesia/5/2005感染后的第1天和Shenzhen/406H/2006感染后的第3天,13D4嵌合抗體的治療效果欠佳。 總之,通過(guò)以上方法我們成功構(gòu)建了四株高致病性禽流感H5N1人-鼠嵌合抗體,它們保留了親本鼠抗的廣譜中和活性,將降低治療人感染疾病時(shí)的HAMA反應(yīng),為治療性抗體在人感染H5N1領(lǐng)域的應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Highly pathogenic avian influenza (Avian influenza AI) is a highly infectious disease caused by influenza A virus H5N1. In recent years with the continuous spread of disease, avian influenza virus not only around the poultry industry suffered huge losses, but also continue to break the species barrier caused by the incident. People infected with highly pathogenic avian influenza face the problem is the high mortality and the lack of effective drug treatment. Due to the continued rapid variation of influenza virus, the H5N1 virus has evolved in nature Mutation Strains antigenically distinct variety of Asia were popular situation, to meet the long-term strategic reserves of drug demand, H5N1 treatment of drug development must have broad spectrum.
Neutralizing antibodies is an effective means of prevention and treatment of viral diseases, but because of the bird flu virus gene mutation frequency is high, especially the hemagglutinin (HA) gene, thus neutralizing monoclonal antibodies against avian influenza virus and narrow spectrum, and only few strains, greatly limits the application of neutralizing antibodies.
Our laboratory recently constructed a hemagglutination inhibition (AI) based on H5 specific monoclonal antibody library activity (in 41 strains of H5N1 contains all 10 representative strains of these genetic clades of monoclonal antibody and the activity of HI in mice as an animal model of neutralizing activity were determined), this study selected four broad spectrum neutralizing monoclonal antibody: the 4D1,10F7,8H5,13D4 humanized reconstruction, in order to reduce the resistance in the treatment of HAMA in the reaction process, lay the foundation for the clinical application of these antibodies as soon as possible; at the same time also hope to establish a therapeutic antibody humanized research platform system, including gene expression vector transformation the construction of efficient and stable expression in mammalian cells, serum-free culture in large scale, antibody purification process.
This study first with RT-PCR from murine hybridoma cells within the variable region gene of four strains of monoclonal antibodies, combined with the constant region of human antibody IgG1, inserted into the pcDNA3.1 expression vector of chimeric antibody products obtained by HI, ELISA, instantaneous; verify the antibody immunofluorescence method with activity in the short term. In order to accumulate certain products for further study, we used site-specific integration expression system FLP-IN, characteristics of the FRT site-specific integration of the antibody to stable high expression after cell screening process, we obtained three strains of stable expression cell lines, expression and antibody production was increased by 3-4 times. In a serum-free medium to obtain antibody purified after us with H5N1 virus strain HI and in vitro experiments, broad-spectrum neutralization test verified chimeric antibody neutralizing activity, and to determine the expression of the entire transformation process of antibody variable region of no activity It has been affected.
This research chooses 13D4 chimeric antibody for large-scale expression and purification. The purification and to meet the needs of mass production, we have no culture method of serum on CHO cells were also explored, including the optimization of serum-free medium nutrient medium, large scale roller bottle culture operation. We found that the addition of BSA could significantly improve the chimeric antibody production, especially the proportion of 3%, the antibody yield increased 8 times, but at the same time the interference brought antibody purification, should consider antibody purity and yield to determine the optimal solution; protein A affinity chromatography was a specific and effective way for antibody purification, we explored different elution PH buffer, pH elution for obtained chimeric antibody purification, the final purity reached 95%, the yield is 90%.
According to the four strains of H5N1 representative of the animal protection experiment results show that in the first and third days after Vietnam/1194/2005 infection, as well as in the BH Goose/Qinghai/15c/2005 and Shenzhen/406H/2006 infection after first days, 13D4 had 100% chimeric antibody treatment. In third days after Indonesia/5/2005 infection and first days after Shenzhen/406H/2006 infection, poor 13D4 chimeric antibody treatment.
In conclusion, through the above method we successfully constructed the chimeric antibody against four strains of highly pathogenic avian influenza H5N1 human mouse, they retained broad-spectrum parental mouse neutralizing activity, will reduce the treatment of human HAMA infection during the reaction, the foundation is set for application in the foundation of human infection with the H5N1 field of therapeutic antibodies.

【學(xué)位授予單位】：廈門(mén)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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本文編號(hào)：1644059