本文選題：A型肉毒毒素 + 中和抗體��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年博士論文

【摘要】： 肉毒毒素是由肉毒梭狀桿菌產(chǎn)生的一種神經(jīng)毒素,分為A-G 7種血清型,是目前已知毒性最強(qiáng)的細(xì)菌蛋白質(zhì),易被某些國家或組織用作生物武器和制造生物恐怖。肉毒毒素中毒治療藥物的研究無論對于治療散發(fā)的民間中毒事件還是預(yù)防生物恐怖襲擊均具有重要意義。目前用于肉毒毒素中毒治療的主要為馬源的血清抗毒素,但馬源抗毒素受供體馬數(shù)量的限制,能提供的血清是有限的,并且對于人而言,它不僅屬于異源蛋白,具有高免疫原性,易引起一些副作用,而且存在潛在病毒污染等問題,應(yīng)用受到很大限制。 作為馬源血清抗毒素的更新?lián)Q代產(chǎn)品,重組基因工程中和抗體可以為肉毒毒素中毒提供有效的預(yù)防和治療手段,是肉毒毒素中毒預(yù)防和治療研究的主要研究方向。本研究的主要目的就是從本實(shí)驗(yàn)室構(gòu)建的大容量全合成人噬菌體抗體庫中篩選、制備A型肉毒毒素的人源中和抗體,為研制A型肉毒毒素中和抗體藥物奠定基礎(chǔ)。 全序列優(yōu)化合成了A型肉毒毒素保護(hù)性抗原Hc(BoNT/A-Hc)基因,并在大腸桿菌實(shí)現(xiàn)了高效可溶性表達(dá),目的蛋白主要以可溶性的方式存在于超聲碎菌上清中,占碎菌上清總蛋白的36-53%。經(jīng)一步純化以后,純度可達(dá)95%以上,得率在30mg/L以上,表達(dá)水平是目前國際報(bào)道的最高水平。 以表達(dá)純化的BoNT/A-Hc蛋白作為抗原,對庫容量為1.35×10~(10)的大容量全合成人源噬菌體單鏈抗體庫進(jìn)行篩選,共鑒定1000多個(gè)克隆,70%左右為陽性克隆。經(jīng)特異性鑒定,約73%陽性克隆特異性好。對其中18株陽性克隆進(jìn)行測序,得到5株具有不同序列的單鏈抗體,它們的重鏈框架均為H5,輕鏈框架均為λ3。建立了5株單鏈抗體的大腸桿菌分泌表達(dá)系統(tǒng),在大腸桿菌中獲得了分泌表達(dá),表達(dá)的目的蛋白保持了與噬菌體抗體相同的特異性。此外,采用競爭ELISA的方法檢測了這5株單鏈抗體與篩選到的其它抗體的抗原表位基本一致。 從這5株陽性克隆中選取了一株親和力較高(KD=7.8×10~(-9)M)的單鏈抗體C10,通過替換抗體輕重鏈可變區(qū)之間的連接肽,將C10單鏈抗體改造成雙價(jià)的Diabody形式抗體,在大腸桿菌KS1000(DE3)中獲得了分泌表達(dá)。純化后的C10 Diabody保持了與抗原結(jié)合的特異性,并且比單鏈抗體更加穩(wěn)定,在4℃保存1周后蛋白基本沒有降解,抗體活性未見明顯下降。雙價(jià)的C10 Diabody與抗原的親和常數(shù)KD為3.57×10~(-10)M,與單鏈抗體相比,其親和力提高了約20倍。此外,還構(gòu)建了C10 IgG4全抗體的輕重鏈表達(dá)載體,并在FreeStyle~(TM)293-F細(xì)胞中的獲得了瞬時(shí)表達(dá),表達(dá)量可達(dá)20mg/L。C10 IgG4全抗體保持了與抗原BoNT/A-Hc特異性結(jié)合的活性,并且穩(wěn)定性有了更大的提高,在4℃保存1個(gè)月后抗體活性未見明顯下降。利用BiaCore測定了C10 IgG4全抗體與抗原的親和常數(shù)KD為6.46×10~(-11)M,與單鏈抗體相比,親和力提高了約100倍。 此外,針對肉毒毒素與受體細(xì)胞結(jié)合的雙受體多表位的特點(diǎn),必須研制多株具有不同表位的抗體進(jìn)行聯(lián)合使用,才能達(dá)到完全封閉結(jié)合表位的作用,從而起到較好的中和保護(hù)效果。本研究采用夾心篩選及固相競爭篩選的方法篩選了與C10具有不同抗原表位的抗體,最終獲得了7株與C10具有不同抗原表位的噬菌體抗體,經(jīng)測序,這7株抗體具有3種不同的抗體基因序列,將這3株抗體改造為單鏈抗體和全抗體,分別在大腸桿菌中進(jìn)行分泌表達(dá)和在FreeStyle~(TM)293-F細(xì)胞中進(jìn)行了瞬時(shí)表達(dá)后,只有2株抗體1B6、2G4保持了與BoNT/A-Hc結(jié)合的活性,且這2株抗體具有不同的抗原表位。采用非競爭酶免疫實(shí)驗(yàn)法測定了1B6、2G4全抗體的親和常數(shù)KD分別為2.38×10-8M和3.99×10~(-8)M。 為了進(jìn)一步分析C10這株單克隆抗體與抗原的結(jié)合表位,設(shè)計(jì)并構(gòu)建了5條BoNT/A-Hc的分段抗原表位肽段,并檢測了C10單克隆抗體與各表位肽段的結(jié)合情況,結(jié)果表明C10的抗原表位位于BoNT/A-Hc 1200-1223位氨基酸之間,而1B6的抗原表位不位于第1200-1223位氨基酸之間,從另一角度證實(shí)了1B6這株抗體與C10具有不同的抗原表位。C10抗原表位與所報(bào)道的已知抗原表位均不相同,新表位的發(fā)現(xiàn)可以使A型肉毒毒素表位繪制圖譜更加完善。 采用細(xì)胞免疫熒光法檢測了C10、1B6和2G4全抗體的體外中和活性。結(jié)果顯示C10全抗體、1B6全抗體和2G4全抗體在體外均能有效地抑制抗原BoNT/A-Hc與NGF誘導(dǎo)分化的受體神經(jīng)細(xì)胞PC-12的結(jié)合,說明C10、1B6和2G4這三株單克隆抗體均具有一定的體外中和活性。將毒素與50μg抗體或抗體混合物,在體外孵育0.5h后,小鼠腹腔注射進(jìn)行體內(nèi)攻毒保護(hù)實(shí)驗(yàn),結(jié)果顯示:1B6抗體單獨(dú)使用時(shí),可以延長小鼠致死時(shí)間,但不能完全保護(hù)20LD50劑量的毒素攻擊;C10抗體單株使用時(shí),可以部分保護(hù)小鼠20LD50劑量的毒素攻擊;2G4抗體單獨(dú)使用和任兩株抗體配對使用時(shí)均能夠完全保護(hù)小鼠20LD50劑量的毒素攻擊;而三株抗體聯(lián)合使用時(shí)更可以完全保護(hù)100LD50劑量的毒素攻擊,即每1mg混合抗體至少能夠完全中和2000LD50劑量的毒素。證明三個(gè)抗體聯(lián)合使用具有良好的體內(nèi)中和保護(hù)效果。這三株抗體均為人源抗體,可直接應(yīng)用于人體使用,而無需人源化改造。 通過以上的研究,從大容量全合成人噬菌體抗體庫中共獲得了3個(gè)針對不同抗原表位的具有內(nèi)體外中和活性的抗A型肉毒毒素的人源單克隆抗體C10、1B6和2G4,為下一步深入研制A型肉毒毒素中和抗體藥物奠定了良好的基礎(chǔ)。
[Abstract]:Botulinum toxin, a neurotoxin produced by Clostridium botulinum, is divided into 7 serotypes of A-G. It is the most toxic protein known at present. It is easily used as a biological weapon and bioterrorism by some countries or tissues. Research on the treatment of botulism in the treatment of botulism is not only for the treatment of sporadic folk poisoning or prevention. Biological terrorist attacks are of great significance. At present, the main treatment of botulism is serum antitoxin of Ma source. However, Ma source antitoxin is limited by the number of donor horses. The serum is limited, and for people, it not only belongs to the heterologous protein, has high immunogenicity, and causes some side effects, but also exists in the presence of some side effects. The application of virus pollution is limited.
As a renewal product of antitoxin of horse source serum, recombinant gene engineering neutralization antibody can provide effective prevention and treatment for botulism, and it is the main research direction in the study of botulism prevention and treatment. The main purpose of this study is to construct large volume full adult phage antibody from this laboratory. The human neutralizing antibody of botulinum toxin type A was prepared by screening in the library, which laid the foundation for developing the neutralizing antibody against botulinum toxin type A.
The whole sequence was optimized to synthesize the A type botulinum toxin protective antigen Hc (BoNT/A-Hc) gene, and the high efficiency soluble expression was realized in Escherichia coli. The target protein mainly existed in the sonographic supernatant in soluble mode. After one step purification, the purity of the total protein of the total protein of the broken bacteria was more than 95%, and the yield was above 30mg/L. The level is the highest level of international reports at present.
The purified BoNT/A-Hc protein was expressed as an antigen, and a large capacity full adult phage single chain antibody library with a capacity of 1.35 * 10~ (10) was screened. More than 1000 clones were identified and about 70% were positive clones. Through specificity identification, about 73% positive clones were specific. 18 positive clones were sequenced and 5 were not. The same sequence of single chain antibodies, their heavy chain frames are all H5, the light chain framework is lambda 3. to establish a 5 single chain antibody of Escherichia coli secretory expression system, the secretory expression in Escherichia coli, the expression of the target protein to maintain the same specificity with phage antibodies. In addition, the use of competitive ELISA method to detect the 5 single strands The antibody epitopes were basically consistent with those of other antibodies screened.
A single chain antibody C10 with high affinity (KD=7.8 * 10~ (-9) M) was selected from these 5 positive clones. The C10 single chain antibody was transformed into a bivalent Diabody form antibody by replacing the connective peptide between the variable region of the weight chain of the antibody, and the secretory expression was obtained in the Escherichia coli KS1000 (DE3). The purified C10 Diabody kept the antigen junction with the antigen junction. It was more specific and more stable than single chain antibody. After 1 weeks of preservation, the protein was basically not degraded and the antibody activity was not significantly decreased. The affinity constant KD of bivalent C10 Diabody and antigen was 3.57 x 10~ (-10) M, and its affinity increased about 20 times compared with single chain antibody. In addition, the weight chain expression of C10 IgG4 full antibody was also constructed. The vector was expressed instantaneously in FreeStyle~ (TM) 293-F cells, and the expression amount could reach 20mg/L.C10 IgG4 full antibody to the activity of specific binding to the antigen BoNT/A-Hc, and the stability was improved greatly. The antibody activity was not significantly decreased after 1 months of preservation at 4 C. BiaCore was used to determine the C10 IgG4 full antibody and antigen. The affinity constant KD is 6.46 x 10~ (-11) M, and its affinity is about 100 times higher than that of scFv.
In addition, in view of the characteristics of bis receptor multiple epitopes binding to botulinum toxin and receptor cells, it is necessary to develop multiple antibodies with different epitopes to be combined in order to achieve the effect of completely closed binding epitopes and thus play a better role in neutralization and protection. This study selected the method of sandwich screening and solid phase competition screening with C10 The antibodies with different epitopes were finally obtained from 7 phage antibodies with different epitopes of C10. After sequencing, the 7 antibodies had 3 different antibody gene sequences, and the 3 antibodies were transformed into single chain and full antibodies. The secreting and expression of the antibodies in Escherichia coli and in FreeStyle~ (TM) 293-F cells were carried out. After transient expression, only 2 antibody 1B6,2G4 maintained the activity of binding with BoNT/A-Hc, and the 2 antibodies had different epitopes. The affinity constant KD of 1B6,2G4 total antibody was 2.38 * 10-8M and 3.99 x 10~ (-8) M., respectively, by non competitive enzyme immunoassay.
In order to further analyze the binding epitopes of C10 monoclonal antibody and antigen, 5 BoNT/A-Hc fragment epitopes were designed and constructed, and the binding of C10 monoclonal antibodies to the peptide segments of each epitope was detected. The results showed that the epitopes of C10 were located between the BoNT/A-Hc and the amino acids of BoNT/A-Hc, and the epitopes of 1B6 were not located. Between 1200-1223 amino acids, it was confirmed from another point of view that the antigen epitopes of the 1B6 strain and C10 were different from the known epitopes of the known antigen, and the discovery of the new epitopes could make the mapping of A type botulinum toxin epitopes more perfect.
The neutralization activity of C10,1B6 and 2G4 total antibody in vitro was detected by cell immunofluorescence. The results showed that all antibody of C10, 1B6 and 2G4 could effectively inhibit the binding of BoNT/A-Hc and NGF induced receptor nerve cell PC-12 in vitro, indicating that all three monoclonal antibodies of C10,1B6 and 2G4 are in vitro. Neutralization activity. After incubating the toxin with 50 mu G antibody or antibody and incubating for 0.5h in vitro, the mice were injected intraperitoneally to protect the toxin in vivo. The results showed that when the 1B6 antibody was used alone, the lethal time of mice could be prolonged, but the dose of 20LD50 could not be completely protected. When the C10 antibody was used alone, the mice could partially protect the 20LD5 of mice. 0 dose of toxin attack; 2G4 antibody alone and two strains of antibody can fully protect the mouse 20LD50 dose of toxin attack; while the three antibody combined use can fully protect the 100LD50 dose of the toxin attack, that is, each 1mg mixed antibody can at least complete the total neutralization 2000LD50 dose of toxin. Prove three antibodies. The combined use has good in vivo and neutralizing protective effects. These three antibodies are human antibodies, which can be directly applied to human body without human modification.
Through the above study, 3 human monoclonal antibodies (C10,1B6 and 2G4) of anti A botulinum toxin with different epitopes of different antigen epitopes were obtained from the large capacity full adult phage antibody library, which laid a good foundation for the further development of A type botulinum toxin neutralizing antibody drugs.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 韋娜;王雙;林建波;孫志偉;陳惠鵬;張惟材;;A型肉毒毒素中和抗體S25全抗體的表達(dá)[J];生物技術(shù)通訊;2011年03期

相關(guān)博士學(xué)位論文 前1條

1 韋娜;A型肉毒毒素受體和中和抗體研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2011年

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本文編號：1912290