A型肉毒毒素人源性中和抗体的制备

发布时间：2018-05-20 00:07

本文选题：A型肉毒毒素 + 中和抗体　；参考：《中国人民解放军军事医学科学院》2008年博士论文

【摘要】： 肉毒毒素是由肉毒梭状杆菌产生的一种神经毒素,分为A-G 7种血清型,是目前已知毒性最强的细菌蛋白质,易被某些国家或组织用作生物武器和制造生物恐怖。肉毒毒素中毒治疗药物的研究无论对于治疗散发的民间中毒事件还是预防生物恐怖袭击均具有重要意义。目前用于肉毒毒素中毒治疗的主要为马源的血清抗毒素,但马源抗毒素受供体马数量的限制,能提供的血清是有限的,并且对于人而言,它不仅属于异源蛋白,具有高免疫原性,易引起一些副作用,而且存在潜在病毒污染等问题,应用受到很大限制。作为马源血清抗毒素的更新换代产品,重组基因工程中和抗体可以为肉毒毒素中毒提供有效的预防和治疗手段,是肉毒毒素中毒预防和治疗研究的主要研究方向。本研究的主要目的就是从本实验室构建的大容量全合成人噬菌体抗体库中筛选、制备A型肉毒毒素的人源中和抗体,为研制A型肉毒毒素中和抗体药物奠定基础。全序列优化合成了A型肉毒毒素保护性抗原Hc(BoNT/A-Hc)基因,并在大肠杆菌实现了高效可溶性表达,目的蛋白主要以可溶性的方式存在于超声碎菌上清中,占碎菌上清总蛋白的36-53%。经一步纯化以后,纯度可达95%以上,得率在30mg/L以上,表达水平是目前国际报道的最高水平。以表达纯化的BoNT/A-Hc蛋白作为抗原,对库容量为1.35×10~(10)的大容量全合成人源噬菌体单链抗体库进行筛选,共鉴定1000多个克隆,70%左右为阳性克隆。经特异性鉴定,约73%阳性克隆特异性好。对其中18株阳性克隆进行测序,得到5株具有不同序列的单链抗体,它们的重链框架均为H5,轻链框架均为λ3。建立了5株单链抗体的大肠杆菌分泌表达系统,在大肠杆菌中获得了分泌表达,表达的目的蛋白保持了与噬菌体抗体相同的特异性。此外,采用竞争ELISA的方法检测了这5株单链抗体与筛选到的其它抗体的抗原表位基本一致。从这5株阳性克隆中选取了一株亲和力较高(KD=7.8×10~(-9)M)的单链抗体C10,通过替换抗体轻重链可变区之间的连接肽,将C10单链抗体改造成双价的Diabody形式抗体,在大肠杆菌KS1000(DE3)中获得了分泌表达。纯化后的C10 Diabody保持了与抗原结合的特异性,并且比单链抗体更加稳定,在4℃保存1周后蛋白基本没有降解,抗体活性未见明显下降。双价的C10 Diabody与抗原的亲和常数KD为3.57×10~(-10)M,与单链抗体相比,其亲和力提高了约20倍。此外,还构建了C10 IgG4全抗体的轻重链表达载体,并在FreeStyle~(TM)293-F细胞中的获得了瞬时表达,表达量可达20mg/L。C10 IgG4全抗体保持了与抗原BoNT/A-Hc特异性结合的活性,并且稳定性有了更大的提高,在4℃保存1个月后抗体活性未见明显下降。利用BiaCore测定了C10 IgG4全抗体与抗原的亲和常数KD为6.46×10~(-11)M,与单链抗体相比,亲和力提高了约100倍。此外,针对肉毒毒素与受体细胞结合的双受体多表位的特点,必须研制多株具有不同表位的抗体进行联合使用,才能达到完全封闭结合表位的作用,从而起到较好的中和保护效果。本研究采用夹心筛选及固相竞争筛选的方法筛选了与C10具有不同抗原表位的抗体,最终获得了7株与C10具有不同抗原表位的噬菌体抗体,经测序,这7株抗体具有3种不同的抗体基因序列,将这3株抗体改造为单链抗体和全抗体,分别在大肠杆菌中进行分泌表达和在FreeStyle~(TM)293-F细胞中进行了瞬时表达后,只有2株抗体1B6、2G4保持了与BoNT/A-Hc结合的活性,且这2株抗体具有不同的抗原表位。采用非竞争酶免疫实验法测定了1B6、2G4全抗体的亲和常数KD分别为2.38×10-8M和3.99×10~(-8)M。为了进一步分析C10这株单克隆抗体与抗原的结合表位,设计并构建了5条BoNT/A-Hc的分段抗原表位肽段,并检测了C10单克隆抗体与各表位肽段的结合情况,结果表明C10的抗原表位位于BoNT/A-Hc 1200-1223位氨基酸之间,而1B6的抗原表位不位于第1200-1223位氨基酸之间,从另一角度证实了1B6这株抗体与C10具有不同的抗原表位。C10抗原表位与所报道的已知抗原表位均不相同,新表位的发现可以使A型肉毒毒素表位绘制图谱更加完善。采用细胞免疫荧光法检测了C10、1B6和2G4全抗体的体外中和活性。结果显示C10全抗体、1B6全抗体和2G4全抗体在体外均能有效地抑制抗原BoNT/A-Hc与NGF诱导分化的受体神经细胞PC-12的结合,说明C10、1B6和2G4这三株单克隆抗体均具有一定的体外中和活性。将毒素与50μg抗体或抗体混合物,在体外孵育0.5h后,小鼠腹腔注射进行体内攻毒保护实验,结果显示:1B6抗体单独使用时,可以延长小鼠致死时间,但不能完全保护20LD50剂量的毒素攻击;C10抗体单株使用时,可以部分保护小鼠20LD50剂量的毒素攻击;2G4抗体单独使用和任两株抗体配对使用时均能够完全保护小鼠20LD50剂量的毒素攻击;而三株抗体联合使用时更可以完全保护100LD50剂量的毒素攻击,即每1mg混合抗体至少能够完全中和2000LD50剂量的毒素。证明三个抗体联合使用具有良好的体内中和保护效果。这三株抗体均为人源抗体,可直接应用于人体使用,而无需人源化改造。通过以上的研究,从大容量全合成人噬菌体抗体库中共获得了3个针对不同抗原表位的具有内体外中和活性的抗A型肉毒毒素的人源单克隆抗体C10、1B6和2G4,为下一步深入研制A型肉毒毒素中和抗体药物奠定了良好的基础。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【引证文献】