本文针对天然百日咳粘着素（natural pertactin，nPRN）和重组百日咳粘着素（recombinant pertactin，rPRN）的制备难点，从分子结构特点出发，设计了nPRN的双离子交换色谱（dual-IEC）纯化策略和rPRN变性蛋白的瞬间稀释复性方法，并通过与百白破联合疫苗（DTaP）的另外两种抗原DTd或TTd的偶联，增强抗原的免疫原性。主要创新点如下：（1）采用GRASP2软件对nPRN进行表面静电势分布分析。结果显示线性分子的两端各具有一个主要的电势区，并且分别荷正电与负电。本文基于nPRN蛋白表面电荷分布相对集中且正负荷电区域位置相对独立的特征，设计了双离子交换色谱（dual-IEC）的纯化策略，包括一步阴离子交换和一步阳离子交换色谱，从百日咳杆菌的热浸提液中分离nPRN。两步色谱依次实现蛋白纯度从1.7%提高至14.6%，以及进一步至33%，收率分别为80%和83%。最终通过一步疏水作用色谱精制后获得的产物纯度达到96%。三步色谱的总收率可以达到61%。（2）rPRN以包涵体的形式在大肠杆菌中实现了高效表达。通过逐滴稀释的方式进行包涵体复性时，75%以上的可溶性产物为聚集体。针对复性过程中大量产生聚集体的问题，我们分析认为聚集形成机制是部分折叠的中间体的C端具有一个强的疏水性内核，能够与新加入的变性蛋白的同一区域发生结合缠绕，导致不同折叠进程的蛋白之间发生聚集。根据上述机理，本文设计了瞬间稀释法，通过将变性蛋白溶液一次性加入复性缓冲液并快速搅拌混匀，提高复性过程的同步性，抑制聚集体的形成。在5L规模的复性条件下，瞬间稀释法的复性收率可以达到70%以上。聚集体和杂质能够通过一步金属螯合色谱同时除去。获得的单体rPRN纯度达到98%以上，总收率约59%，从1L发酵液中获得了320 mg rPRN蛋白。（3）将rPRN共价偶联至百白破联合疫苗（DTaP）的另外两种抗原DTd或TTd上。等电点滴定、SDS-PAGE等方法分析显示类毒素蛋白表面氨基基团已被封闭，因此交联反应可以通过温和的EDC/sNHS介导的载体蛋白上的羧基基团反应实现。偶联产物与原蛋白的分离通过一步金属螯合色谱实现。SDS-PAGE和高效液相色谱分析显示产物纯度达到90%以上，其中单价偶联物的含量>65%。圆二色谱以及内源性荧光光谱分析显示偶联产物保留了两种原蛋白的高级结构。免疫原性的评价显示rPRN连接至DTd获得的偶联抗原能够增强Th1及Th2型免疫响应，而与TTd连接则主要显示了增强的Th1型响应。本文还对百日咳菌毛血清型3亚基（rfim3）进行了重组表达和纯化。包涵体蛋白质的复性收率达到50%以上。经金属螯合色谱结合凝胶过滤色谱进行纯化。最终产物纯度>99%，总收率30%。1L发酵液中约可以获得33 mg rfim3蛋白。;To overcome the difficulties in preparation of natural pertactin (nPRN) and recombinant pertactin (rPRN) for production of acellular pertussis vaccine (aPV), we developed a “dual-IEC” strategy for purification of nPRN from Bordetella pertussis and a flash-batch dilution method for refolding of rPRN inclusion bodies expressed in E. coli based on its structural characteristics. Besides, rPRN was conjugated with either diphtheria toxoid or tetanus toxoid. The resultant conjugates revealed an enhanced immunogenicity. The main innovations are as follows:(1) We performed the surface potential analysis with GRASP2 program for rPRN. The results demonstrated that there are two major charge patches, one negative and one positive, which are located separately on this linear protein. For this special feature, we designed a dual ion exchange chromatography (dual-IEC) strategy including an anionic exchange and a cationic exchange process for separation of natural pertactin from the heat extract of Bordetella pertussis. The initial anionic exchange chromatography concentrated the product from 1.7 to 14.6%, with recovery of 80%. The second cationic exchange chromatography increased the purity to 33%, with recovery of 83%. The final purification was accomplished by hydrophobic interaction chromatography, yielding a purity of 96%. The overall recovery of the three columns was 61%.(2) Recombinant pertactin was highly expressed in the form of inclusion bodies in E. coli. However, up to 75% of the protein turned out as aggregates when refolding by pulse-fed batch dilution. The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein, resulting in aggregation between proteins with different folding states. The key factor for prevention of aggregate formation was to improve the synchronization of refolding. For this purpose, flash-batch dilution was conducted in a way of one-shoot feed and immediate mixing at a 5 L scale, achieving a refolding yield about 70%. The remaining aggregates were efficiently removed along with impurities by one-step chromatography of Ni-resin. The purity of monomeric pertactin was >98%. An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%. (3) Conjugation of rPRN to diphtheria toxoid (DTd) or tetanus toxoid (TTd) was attempted to enhance the immunogenicity. The cross-linking reaction was mediated by EDC/sNHS in a mild way since the amino groups on the protein surface have been blocked as demonstrated by analyses of SDS-PAGE and pI titration. Separation of the conjugates from parent proteins was achieved by a one-step chromatography on Ni-resin. SDS-PAGE and high-performance size exclusion chromatography (HP-SEC) analyses showed the conjugates with > 90% purity, among which mono-valent conjugates accounted for > 65%. Characterization by circular dichroism and fluorescence displayed that the conjugates conserved an advanced structure of both PRN and the carrier proteins. The evaluation of the immunogenicity was performed on NIH mice. The results with respect to PRN-specific antibody levels, ex vivo cytokines release and GC B cell proliferation showed that conjugation of PRN to DTd led to improving mixed Th1/Th2 responses related to simply mixing, and that of PRN to TTd showed an increase mainly in the Th1 response.Besides, recombinant fimbriae serotype 3 (fim3) was highly expressed in the form of inclusion bodies in E. coli. Optimized refolding condition was determined and yielded a refolding recovery of > 50%. Purification was achieved through immobilized metal affinity chromatography combing size exclusion chromatography. A final product with a purity of > 99% and an overall recovery of 30% were obtained, yielding about 33 mg rfim3 from 1L fermentation liquor.