脊髓灰质炎病毒C3表位和C-myc十肽表位在细菌表面的表达

为了进一步验证载体pCSB136和pCSX72作为表面呈现载体的可行性 ,化学合成脊髓灰质炎病毒C3表位和C myc十肽表位的基因片段 ,并插入到上述载体的相应位点间 ,采用全菌PCR筛选到正向插入的重组子 ,全细胞ELISA和电镜观察显示 ,重组蛋白以杂和菌毛的形式得到了表达 ,并保持CS3和外源表位的抗原性 ,表明脊髓灰质炎病毒C3表位和C myc十肽表位在细菌表面得到了表达 ,证实载体pCSB136和pCSX72可用于外源表位的呈现 ,为构建基因工程活菌疫苗奠定了基础     

脊髓灰质炎病毒C3表位和C—myc＋肽表位在细菌表面的表达

为了进一步验证载体pCSB136和pCSX72作为表面呈现载体的可行性,化学合成脊髓灰质炎病毒C3表位和C-myc十肽有位的基因片段,并插入到上述载体的相应位点间,采用全菌PCR筛选到正向插入的重组子,全细胞ELISA和电镜观察显示,重组蛋白以杂和菌毛的形式得到了表达,并保持CS3和外源位的抗原性,表明脊髓灰质炎病毒C3表位和C-myc十肽捕位在细菌表面得到了表达,证实载体pCSB136和pCSX72可用于外源表位的呈现,为构建基因工程活苗疫苗奠定了基础。     

大肠杆菌耐热肠毒素在细菌表面的呈现

为了探讨CS3菌毛呈现载体用于呈现立体表位的可行性,选择大肠杆菌耐热肠毒素（ST）作为靶蛋白,通过全细胞ELISA,电镜技术来检测重组蛋白的表达,结果显示重组蛋白以杂合菌毛的形式得到了表达,并保持有CS3载体蛋白的抗原性,初步表明ST在细菌表面得到呈现,CS3菌毛呈现载体可用于立体表位的呈现。     

CS3菌毛可以作为异源抗原决定簇的表达载体

CS3是肠毒素源性大肠杆菌的菌毛蛋白 ,是一种很强的免疫原 .利用CS3菌毛作为异源抗原决定簇载体 ,在计算机分析预测CS3亚基的抗原表位区、二级结构的基础上 ,运用PCR定点突变在CS3亚基结构基因中引入了SacⅡ酶切位点序列 ,插入霍乱毒素B亚基抗原表位CTP3的DNA序列 ,构建了表达CS3/CTP3的重组菌株 .电镜和免疫电镜观察证明 ,CS3/CTP3以杂合菌毛的形式存在于菌体表面 .SDS聚丙烯酰胺凝胶电泳显示了CS3/CTP3杂合蛋白的存在 .口服和腹腔注射免疫Balb/c小鼠 ,该重组菌株可诱发抗CS3和抗CTP3的双重免疫应答 .结果表明CS3可以作为表达异源抗原决定簇的表达载体 ,可望成为研制口服粘膜免疫多价疫苗的新型系统     

CS3菌毛可以作为异源抗原决定簇的嵌合表达载体

CS3是肠毒素源性大肠杆菌的菌毛蛋白,是一种很强的免疫原。研究了利用CS3菌毛作为异源抗原决定簇载体的可能性。在计算机分析预测CS3亚基的抗原表位区、二级结构的基础上,运用PCR定点突变在CS3亚基结构基因中引入了SacⅡ酶切位点序列,插入编码霍乱毒素B亚基抗原表位CTP3的DNA序列,构建了表达CS3/CTP3的重组菌株。电镜和免疫电镜观察证明,CS3/CTP3以杂合菌毛的形式存在于菌体表面,SDS聚丙烯酰胺凝胶电泳显示了CS3/CTP3杂合蛋白的存在。口服和腹腔注射免疫Bal b/c小鼠,该重组菌株可诱发抗CS3和抗CTP3的双重免疫应答。结果表明CS3可以作为表达异源抗原决定簇的表达载体,可望成为研制口服粘膜免疫多价疫苗的新型系统。     

2型猪链球菌菌毛结构亚蛋白tSSU0473的原核表达与免疫学特性研究

目的：探究2型猪链球菌（S.suis2）强毒株05ZYH33的srtF基因簇编码的菌毛结构亚蛋白SSU0473的细菌定位及其免疫保护效能。方法：原核表达截短的SSU0473（tSSU0473）,并以亲和层析法纯化目的蛋白,Western印迹检测tSSU0473蛋白的免疫原性,ELISA法检测多抗血清的效价及IgG亚型,小鼠试验测试重组蛋白的免疫保护效能,免疫电镜观测tSSU0473蛋白的细菌定位。结果：在原核系统中表达了tSSU0473蛋白;ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体;动物试验表明tSSU0473蛋白免疫小鼠可抵御致死剂量病原体的攻击,显示出较好的免疫保护作用;免疫电镜检测显示tSSU0473蛋白定位于细菌表面。结论：菌毛亚蛋白tSSU0473是S.suis2膜表面蛋白,具有良好的免疫原性和免疫保护性,可作为S.suis2亚单位疫苗的候选分子。研究结果为系统揭示S.suis2的菌毛生物学结构与功能奠定了基础。     

CTB/CS3融合蛋白与GM1结合能力和免疫原性的分析

免疫霍乱毒素B亚单位（CTB）或肠毒素大肠杆菌（ETEC）定居因子CS3可使人体对ETEC的侵染有保护作用.为探索研制ETEC双组分亚单位疫苗的可行性,利用大肠杆菌诱导表达系统表达了CTB与CS3的融合蛋白（CTB/CS3）.蛋白质印迹结果表明,诱导表达的29 ku蛋白具有CTB和CS3蛋白双重抗原性.经Ni-NTA亲和层析纯化获得重组蛋白CTB/CS3,复性的重组蛋白可以部分形成五聚体并保留了与神经节苷脂GM1的结合能力.动物实验表明,融合蛋白CTB/CS3具有CTB和CS3蛋白的双重免疫原性,同时,CTB的免疫载体作用提高了CS3的免疫强度.     

表达轮状病毒SA11株Vp4的抗原表位诱导病毒中和抗体生成

以昆虫病毒Flockhousevirus（FHV）外壳蛋白为载体的外源抗原表位表达系统（FHV-RNA2载体系统）．在重组杆状病毒和重组pET系统中构建和表达了SA11Vp4胰酶切割位点两侧和重叠切割位点3个抗原表位氨基酸序列（抗原表位A,aa223~242;抗原表位B,aa243～262;抗原表位C,aa234～251）,并对其免疫原性进行了研究。结果表明：这3个抗原表位能诱导动物产生抗同源氨基酸序列的抗体和抗同源病毒（SA11）感染性的血清中和抗体。研究结果提示：RVVp4胰酶切割位点区氨基酸序列除了具有胰酶切割增强病毒感染力外,还具有诱导动物机体产生血清中和抗体的能力,是RV重组抗原表位亚单位疫苗研究中重要的抗原表位氨基酸序列。     

转红色荧光蛋白基因唐鱼外源基因拷贝数的测定

目的:测定转红色荧光蛋白基因唐鱼的外源基因拷贝数。方法:以杂合F5代和杂合F6代转红色荧光蛋白基因唐鱼为材料,以其外源插入片段(pDsRed-mylz2)与基因组插入位点5′侧翼区之间的边界序列作为特异性内参片段,同时以外源整合的红色荧光表达载体序列(pDsRed-mylz2)作为目的基因,采用实时荧光定量PCR技术测定外源基因整合的拷贝数。结果:运用外源基因与特异内参在同批次实时荧光定量PCR中的初始拷贝数比值,得到杂合F5代和杂合F6代转红色荧光蛋白基因唐鱼中插入的外源红色荧光表达载体序列pDsRed-mylz2的拷贝数均值均为3。结论:转红色荧光蛋白基因唐鱼的外源基因拷贝数为3。     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin.

Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtained from a wide variety of different vertebrate species. However, none of the new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb are differentially distributed in tissues. All of these MAb immunocytochemically detected epitopes in embryonic chick cartilage and bone marrow. Three of them (4C3, 7D4, and 6C3) recognized epitopes in adult human skin. All three detected epitopes in the epidermis, one (6C3) strongly detected epitopes in the papillary dermis, and two (4C3, 7D4) detected epitopes in the reticular dermis. Immunostaining patterns in skin using the new MAb directed against native CS/DS structures were distinctly different from those obtained using MAb against the common CS isomers. The distribution of these CS epitopes in functionally distinct domains of different tissues implies that these structures have functional and biological significance.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections

In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x10⁶) and provided 80–100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.     

Dipsticks for rapid detection of Plasmodium in vectoring Anopheles mosquitoes

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.     

Flexibility in MHC and TCR recognition: degenerate specificity at the T cell level in the recognition of promiscuous Th epitopes exhibiting no primary sequence homology

Recognition of peptide Ags by T cells through the TCR can be highly specific. In this report we show the degeneracy of Ag recognition at both MHC and TCR levels. We present evidence that unrelated promiscuous Th cell epitopes from various protein sources exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive proliferative responses at the bulk T cell level. This epitopic mimicry was also observed when peptide (CS.T3(378-395) and TT(830-844))-specific CD4+ T cell lines and T cell hybridoma clones were used in proliferation and Ag presentation assays. A scrambled CS.T3(378-395) peptide did not show any proliferation, indicating that the specificity of the cross-reactive responses may be linked with the primary structure of the peptides. Blocking of CS.T3(378-395)-specific CD4+ T cell proliferation by anti-MHC class II mAb showed that recognition of promiscuous T cell epitopes is largely in association with MHC class II molecules. These findings suggest that promiscuous Th epitopes may be useful in designing peptide-based vaccine constructs. At the same time these results show that at the T cell level there may be a great deal of immunological cross-reactivity between heterologous pathogens, and because of this the host's response to a pathogen may be modified by its previous experience with other unrelated pathogens.     

Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.

The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes.     

Dendritic cells and hepatocytes use distinct pathways to process protective antigen from plasmodium in vivo

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2K(b) epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.