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疟疾是一种古老的疾病，也是世界范围内危害最严重的寄生虫病。本文以我国第一个恶性疟原虫多表位嵌合抗原M.RCAg-1（简称M312）研制中的过程工程难点为对象，解决M312分离纯化过程中不稳定的问题，建立大规模纯化M312的工艺，探索如何增强M312免疫原性，为疟疾疫苗的研制提供工程科学基础。论文的主要创新点如下：（1）发现了M312在分离纯化中不稳定的原因并确立了对策。通过加入EDTA和咪唑作为分离纯化伴侣，成功抑制了溶液中宿主大肠杆菌蛋白酶对产物的降解，提高了分离纯化的收率。其中咪唑用于分离纯化伴侣未见报道，其保护产物的机制可能是咪唑环与金属蛋白酶发生配位螯合，与EDTA合用导致金属蛋白酶活性降低或者失活，抑制蛋白酶对M312的破坏。（2）建立了以EDTA和咪唑为分离纯化伴侣、以金属螯合层析与高效凝胶过滤层析为主干的分离纯化M312的工艺，在洁净车间批量制备了克级M312，获得了纯度大于95%的目标蛋白，总收率约为600 mg/L培养基。批量制备的M312内毒素残留、宿主蛋白残留和DNA残留都符合美国FDA标准。质谱显示其分子量与理论值一致，圆二色光谱表征其二级结构主要是无规卷曲。与弗氏佐剂配伍免疫小鼠能产生大量的抗原特异性抗体。此外，纯化的M312能够在4 °C稳定存放超过6个月。（3）尝试了一种增强M312免疫原性的新策略。将M312与截短的鞭毛蛋白（tFL）共价连接，利用M312上有游离半胱氨酸的特点，通过简单温和的SC-PEG-MAL介导的反应，实现了两个蛋白质的偶联。偶联产物M312?PEG?tFL与残留的修饰剂通过一步离子交换层析实现彼此分离，残留的原蛋白通过一步高效凝胶过滤层析移去。SDS-PAGE显示产物纯度约90%，其中单价偶联物的含量约为50%。圆二色光谱显示偶联产物基本保留了两种原蛋白的高级结构。免疫原性评价显示相比于原始的M312抗原，与tFL交联后的抗原产生了更强的体液免疫应答，并产生大量的特异性抗体。交联产物能够在小鼠中诱导脾细胞产生高程度增殖水平，促进淋巴细胞增殖，并产生大量的记忆T细胞。血清中抗体分型和脾细胞分泌的细胞因子分析发现，单独的M312更倾向于Th2型细胞免疫，而与tFL交联后能够促使Th1型细胞免疫。此外，M312?P5k?tFL使小鼠脾细胞产生更多的TNF-α，表明固有免疫应答更有效，同时tFL保留了TLR5的活性。;Malaria is an ancient and infectious disease, is one of the most serious in human populations. This study aimed at solving the problems in downstream processing of a new multi-epitope chimeric antigen of Plasmodium falciparum, M.RCAg-1 (M312 in short), which could be used for development of a malaria vaccine. Research efforts included solving the problem of instability in the separation and purification of M312, establishment of large-scale purification of M312. Also a bioconjugate vaccine based on M312 was prepared to increase the immunogenicity, which provides an engineering foundation for the development of malaria vaccine. Major novelties are as follows:(1) The reasons for the instability of M312 in separation and purification were discovered and the countermeasures were established. By the addition of imidazole and EDTA as chaperone of separation and purification, the degradation of the target protein by the presence of protease(s) in the initial extraction solution after cultivation and disintegration of the E. coli was successfully depressed, and the yield of separation and purification were improved. However, the effect of imidazole as the chaperone has not been reported yet. Its possible mechanism for protection of the target product is the coordinate chelating with metalloprotease(s) through the imidazole ring. Combination with EDTA, imidazole could denature the protease(s) and protect M312 from being degraded.(2) A complete process was designed and established for separation and purification of M312, featuring combination of metal-chelate chromatography and high-performance gel filtration chromatography as major steps. The process was scale-up to produce gram quantity of M312 in sterile clean workshop. The end product reached 95% purity, with 600 mg/L(culture medium) total recovery. The residuals of endotoxin content, host cell proteins, host cell DNA met with the standard of Food and Drug Administration of the US. The structure of M312 was characterized. Mass spectrometry revealed the molecular weight of M312 was consistent with the theoretical value. Circular dichroism indicated that the secondary structure consists of mainly disordered random coil. Immunization of mice with the M312 and Freund's adjuvant could generate large quantity of antigen-specific antibodies. Furthermore, the M312 prepared was stable at 4 °C for up to 6 months.(3) A strategy for enhancement of M312’s immunogenicity was attempted. There exist free cysteine(s), so a conjugate antigen was designated to link a free cystine residual of M312 with an amino group of tFL, by the crosslinker of SC-PEG-MAL to carry out the coupling of the two proteins. The reaction was carried out under mild conditions, and the conjugate, tFL?PEG-MAL, was separated from the residual crosslinker by ion exchange chromatography. And then the final product M312?PEG?tFL was purified by high-performance gel filtration chromatography. SDS-PAGE showed a purity of about 90% of the conjugate product with 50% mono-M312?PEG?tFL conjugate. Circular dichroism revealed the conjugate retaining the secondary structure of M312 and tFL. After boosting immunization in mic, the M312?PEG?tFL elicited much higher anti-M312 antibody titer and stronger humoral immunity than M312 alone. Compared with M312, the M312?PEG?tFL conjugates enhanced the proliferation index, lymphocyte activation and memory T cell generation. IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could trigger the Th1 polarization, while the antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more efficient innate immune response was provoked by the M312?P5k?tFL, as determined by the detection of antigen-specific TNF-α secretion by splenocytes indicating that the M312?P5k?tFLcould elicit more efficient innate immune responses while the tFL in the conjugate retained the function as an agonist of TLR5.
|郭方霞. 新型疟疾嵌合抗原M312的纯化工艺及增强其免疫原性的研究[D]. 中国科学院研究生院,2017.|
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