恶性疟原虫多表位人工随机重组疫苗M.RCAg-1的中试规模制备与鉴定

中试规模高效制备恶性疟原虫多表位人工随机重组疫苗M.RCAg-1,并对其免疫原性进行鉴定。20 L培养基规模发酵培养M.RCAg-1的工程菌,产物经高压匀浆破碎后,通过镍琼脂糖凝胶FF层析和琼葡糖凝胶G200 HP层析两步分离纯化获得中试M.RCAg-1。将15只BALB/c小鼠随机地平均分为3组（弗氏佐剂组、中试M.RCAg-1与弗氏佐剂配伍组和小试M.RCAg-1与弗氏佐剂配伍组）进行皮下免疫,利用间接Elisa和间接免疫荧光（IFA）技术对3次免疫后小鼠血清中的特异性抗体进行分析。IPTG诱导4 h后发酵结束,菌体产量25 g/L,M.RCAg-1约占菌体总蛋白的24%;经两步柱层析分离纯化后,目的蛋白纯度大于95%,回收率高达53.2%;与弗氏佐剂对照组相比,中试M.RCAg-1和小试M.RCAg-1都能在小鼠体内诱生高水平的特异性抗体应答,其抗体滴度均可达到1:1 024 000。与此同时,两实验组鼠血清抗体的对疟原虫天然抗原都有识别,其识别水平并无差异（P>0.05）。本研究成功地高效制备出多表位人工随机重组疫苗M.RCAg-1,并在小鼠体内证实其具有良好的免疫原性,为随后进行的M.RCAg-1的临床前研究奠定基础。     

鼠疟模型作为恶性疟原虫多表位疫苗保护性的探索试验

目的为寻找恶性疟原虫基因工程多表位疫苗保护性评价的模型动物,利用小鼠约氏疟原虫(Plasmodiumyoelii)及小鼠伯氏疟原虫(Plasmodiumberghei)模型对本实验室构建的恶性疟原虫(Plasmodiumfalciparum)多阶段、多表位基因工程候选疫苗进行了体内保护性及免疫模式探索试验。结果在约氏疟原虫模型,先用该疫苗基因的大肠杆菌表达蛋白免疫,后用含同一基因的重组痘苗病毒加强免疫组,与野生型痘苗病毒免疫组及空白对照组相比,小鼠的死亡时间延长(P<0.01)。结果初步显示,约氏疟原虫鼠疟模型可用作该基因工程多表位恶性疟原虫候选疫苗的保护性评价。     

丙型肝炎病毒与恶性疟原虫复合多表位抗原基因在小？…

目的：探讨丙型肝炎病毒（ＨＣＶ）及恶性疟原虫（Ｐｆ）复合ＤＮＡ疫苗的可行性。方法：把ＨＣＶ复合多表位抗原基因ＰＣＸ与Ｐｆ复合基因ＡＢ克隆到带ＣＭＶ启动子的真核表达载体ｐｃＤＮＡ３中,构建真核表达载体ｐｃｄＤＮＡ３／ＣＡＢ,肌肉注射免疫小鼠及家兔,检测其诱发特异性免疫应答水平及安全性。结果：小鼠及家兔分别于免疫后第６周及第８中检测到抗ＧＺ－ＰＣＸ抗体,于第１０周达最高,滴度分别为１：４００及１：３２     

恶性疟原虫多表位蛋白疫苗M.RCAg-1和M.RCAg-3佐剂的配伍

目的 评价恶性疟原虫多表位蛋白疫苗M.RCAg-1和M.RCAg-3与不同佐剂最优配伍方式并进行初步的免疫机制研究.方法 将多表位疫苗表达纯化后混合4种佐剂(AD、ADL、CpG及CpG&A1)经皮下注射BALB/c小鼠,免疫3次,每次间隔2周.测定血清中抗M.RcAg-1和M.RCAg-3抗体滴度、IgG分型及血清抗体对抗原单表位识别.间接免疫荧光检测血清对天然疟原虫的识别.结果 佐剂ADL和AD分别能显著增强M.RCAg-1(P<0.05)和M.RCAg-3(P<0.05)免疫反应.而佐剂CpG和CpG&A1增强抗原免疫反应的能力相对较弱.与M.RCAg-1、M.RCAg-3和CpG、CpG&A1配伍的组相比,AD和ADL佐剂组的抗体水平和间接免疫荧光反应也都显著增强(P<0.05,P<0.05),抗体对单表位识别显著,抗体IgG分型中IgG1和IgG2a水平较高.结论 佐剂ADL和AD的效果显著,可以作为与M.RCAg-1和M.RCAg-3配伍的候选临床应用佐剂.     

重组恶性疟多表位杂合蛋白在大肠杆菌中的表达

将化学合成的恶性疟原虫保护性抗原复合基因 ( HGFSP)与表达载体 p RSET重组并转化大肠杆菌BL2 1 ,工程菌经 IPTG诱导后 ,HGFSP得到高效表达。 SDS- PAGE结果显示表达产物以非融合、可溶性的形式表达 ,分子量为 2 3k Da,占总菌体蛋白的 2 3.65%。Dot- ELISA和 Western- blot分析表明表达产物具有免疫原性。     

含恶性疟原虫Pf70基因片段表达载体DNA的免疫学特性

目的 研究恶性疟原虫Pf70 DNA片段作为DNA疫苗候选抗原基因的可能性。方法 应用PCR方法从含有恶性疟原虫Pf70基因DNA片段的质粒pGEX-Pf70中扩增出包含Pf70 DNA片段的长度为957bP的PCR产物。将其插人带有乙肝表面抗原基因的真核表达载体pCMV-S中，构建出重组质粒pCMV-S-pf70。用提纯的重组质粒pCMV-S-Pf70作为DNA免疫制剂，以质粒pCMV-S DNA和经过谷胱甘肽亲和层析法纯化的融合蛋白GST-Pf70作为对照，免疫昆明种小白鼠。每隔14d加强免疫1次，加强免疫2次后第7天采小鼠全血，用流式细胞仪检测CD8 T淋巴细胞和CD4 T淋巴细胞的水平，以检测体液免疫和细胞免疫状况。结果 接种了重组质粒pCMV-S-Pf70小鼠CD8 T淋巴细胞的绝对量增加，其百分比含量增加了39％；CD4 T淋巴细胞的相对含量保持恒定，绝对量稍有增加。用ELISA法分别检测经重组质粒pCMV-S-Pf70免疫的小鼠和经融合蛋白GST-Pf70免疫的小鼠血清中抗Pt70蛋白质的抗体滴度，发现前者抗体滴度约为1：800，远远低于后者的滴度(1：5000)。研究结果证明，重组质粒pCMV-S-Pf70 DNA可以诱导小鼠产生很强的细胞免疫和相对于蛋白质免疫而言较弱的体液免疫；Pt70蛋白质可以诱导小鼠产生很强的体液免疫。结论 恶性疟原虫红内期Pf70 DNA片段是有前景的红内期DNA疫苗候选抗原基因片段。     

恶性疟原虫多表位疫苗M.RCAg-1在大肠杆菌中的表达和纯化

在大肠杆菌中表达和纯化恶性疟原虫随机重组多表位蛋白疫苗M.RCAg-1,将目的蛋白纯度最高化,回收率最大化。从工程菌BL21(DE3)-M.RCAg-1/pDS-eX提取重组质粒进行测序验证,用IPTG诱导表达,分别采用Ni亲和纯化,阴离子交换层析和凝胶过滤层析等不同组合方式的5种纯化方案进行纯化,根据最终纯度和回收率选择最佳纯化方案。Ni-NTA Agarose特异性好,一步纯化后M.RCAg-1纯度可达79.98%,后续的两步阴离子交换和分子筛纯化能显著提高M.RCAg-1的纯度,最终纯度可超过99%,回收率大于15%。试验确立了恶性疟原虫多表位蛋白疫苗M.RCAg-1在大肠杆菌中的表达和纯化方案,为下游放大生产和进一步研究M.RCAg-1的效果奠定了基础。     

恶性疟疾疫苗M.RCAg-1与候选佐剂纳米乳配伍的免疫原性研究

为了评价纳米乳作为佐剂对恶性疟疾人工重组蛋白疫苗M.RCAg-1免疫原性的影响,将纳米乳佐剂与M.RCAg-1相配伍,于第0、14、28 d肌肉注射免疫小鼠,抗原量为20μg/只。第3次免疫后10 d,眼球取血并取脾细胞。采用ELISA法检测小鼠血清特异性Ig G抗体水平、抗体亚类及其对抗原单表位的识别,用间接免疫荧光反应(IFA)检测抗体对恶性疟原虫天然蛋白的识别,用ELISPOT法检测小鼠脾淋巴细胞免疫应答水平。结果显示疫苗佐剂组小鼠血清中抗M.RCAg-1的抗体水平可达1∶108;疫苗佐剂组产生的特异性Ig G抗体以Ig G1为主;疫苗佐剂组抗体对抗原单表位和天然虫体的识别效果显著。各表位和蛋白诱发免疫小鼠分泌IFN-γ的特异性淋巴细胞克隆数高于分泌IL-4的特异性淋巴细胞克隆数。结果提示纳米乳作为恶性疟疾疫苗M.RCAg-1的佐剂,能够显著提高机体的体液免疫和细胞免疫应答水平。     

我国恶性疟原虫裂殖子表面蛋白MSP-2的抗原表位分析

根据我国恶性疟原虫株MSP-2基因序列,检索我国虫株MSP-2是否具有国外已鉴定的MSP-2抗原表位。结果显示,我国虫株MSP-2具有已鉴定的FC-27等位基因型可变区抗原表位STNS和DTPTATE。同时应用计算机辅助抗原表位分析技术对MSP-2进行抗原表位分析预测,并以合成肽技术鉴定了预测表位的免疫原性。抗原指数分析表明,MSP-2分子亲水性指数和侧链易曲性指数最高的区域分别位于aa153～166和aa65～72,而aa53～60可能是我国虫株特异的抗原表位。用4个合成肽进行免疫学鉴定,证实预测的表位可以诱导抗肽抗体反应,免疫血清可识别重组MSP-2和恶性疟原虫全抗原中MSP-2抗原区带,且合成肽可与我国流行区高度免疫人群免疫血清反应。由于这些表位存在于我国海南、云南、安徽株MSP-2序列中,可以作为研制我国恶性疟重组多价表位疫苗的候选抗原表位。     

恶性疟原虫红内期疫苗的研究现状和展望

恶性疟原虫红内期疫苗的研究现状和展望管惟滨，孙树权（上海第二军医大学寄生虫学教研室，上海２００４３３）疟疾仍然是当今人类最主要的传染病之一，全球每年约有一亿二千万疟疾患者，带虫者达三亿。由于疟原虫的抗药性和媒介按蚊对杀虫剂也产生抗性，因此常用的药物防...     

幽门螺杆菌全长cagA基因和霍乱毒素B亚单位基因的克隆与表达

目的：构建表达幽门螺杆菌（Hp)细胞毒素相关蛋白（CagA)及粘膜免疫佐剂量霍乱毒素B亚单位（CTB）的重组质粒，并在大肠杆菌中表达获得基因重组蛋白。方法：用PCR方法从幽门螺杆菌扩增CagA基因片段，从霍乱弧菌扩增CTB基因片段，将它们转入原核载体质粒pGEMEX-1，在大肠杆菌DH5α中克隆，并在JM109DE3中表达。结果：重组质粒pEGEMEX-CTB的全长序列经分析与GenBank公布的序列相符；各表达蛋白经SDS－PAGE分析，相对分子量与文献相符；重组蛋白经Westem blot检测有较强的抗原性。结论：基因重组菌表达的融合蛋白有可能作为有效抗原用于幽门螺杆菌疫苗的研制及检测试剂盒的制备。     

Prevention of mucosally induced uveitis with a HSP60-derived peptide linked to cholera toxin B subunit

Oral administration of the uveitogenic peptide (aa 336-351) derived from human HSP60 induced clinical and histological manifestations of uveitis in 65.8% (48/73) of Lewis rats. Uveitis was significantly decreased to 16.7% (11/66) in parallel experiments with the peptide linked to recombinant cholera toxin B subunit (rCTB), also given by mouth (chi(2)=34.2, p<0.0001). The protective efficacy between tolerized and immunized animals was 74.7%. Adoptive transfer of mesenteric lymph node cells from tolerized rats prevented the development of uveitis. A significantly higher proportion of regulatory CD4(+)CD45RC(low)RT6(+) subset of Th2 memory cells were found in the mesenteric lymph nodes (p<0.005) and spleens (p相似文献   

Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, where coupling has been obtained either by genetic fusion or chemical conjugation. For genetically fused immunogens both N- and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impaired the biological functions of CTB, such as the ability to form pentamers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has been used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptococcal protein G, which is known to fold independently of N- or C-terminal fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) columns. The CTB fusion proteins were compared for their ability to form pentamers, by gel electrophoresis and size-exclusion chromatography, and it was concluded that all three fusion proteins were able to pentamerize. Interestingly, the C-terminal fusion to CTB showed most efficient pentamerization, while the dual fusion was much less efficient. Purified pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB. In addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM1, and it was found that the C-terminal fusion (CTB-BB) showed significant GM1-binding, but that also the N-terminal and dual CTB fusion proteins bound GM1, although less efficiently. The implications of the results for the design and use of CTB fusion proteins as subunit vaccines are discussed.     

Identification of an immunodominant T cell epitope on cholera toxin

Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal immunogen as well as a strong mucosal adjuvant to related and unrelated antigens. The mucosal immune response to CT is T cell dependent and MHC class II restricted. The epitopes on CT recognized by T cells have not been identified. The purpose of this study was to determine the fine specificity of T cell recognition of both the CT A subunit (CT-A) and the CT B subunit (CT-B) by using a range of synthetic peptides. After immunization with CT-B or CT-A in CFA subcutaneously, the peripheral lymph node T cells were stimulated with different synthetic peptides in vitro The peptide specificity of T cell recognition was identified by assaying T cell proliferation and interleukin-3 production. T cells from C57BL/6 (H-2^b) high responder mice recognized one immunodominant epitope (peptide 89–100) and one weak epitope (peptide 31–50) on CT-B and two epitopes (peptide 21–39 and 180–194) on CT-A. The immunization of C57BL/6 mice with synthetic immunodominant CT-B peptide 89–100 induced T cell immunity to the pentameric CT-B. Induction of tolerance to CTB peptide 89–100 by i.v. injection in high responder C57BL/6 mice induced unresponsiveness to mucosal immunization with CT, compatible with an immunodominant role for this T cell epitope.     

Thoracolumbar sympathetic preganglionic neurons in the dorsal commissural nucleus of the male rat: an immunohistochemical study using retrograde labeling of cholera toxin subunit B

The cell morphology of sympathetic preganglionic neurons (SPNs) in the dorsal commissural nucleus was studied by the retrograde labeling technique using cholera toxin subunit B (CTb) as a tracer. A small amount of an aqueous solution of CTb was injected unilaterally into the major pelvic ganglion of the male rat. Labeled SPNs were detected immunohistochemically using anti-CTb antiserum. Most of the labeled SPNs were observed in L₁ to L₃, and a very small number in T₁₃. They were observed bilaterally in the sympathetic nuclei, such as the intermediolateral cell column, intercalated nucleus and the dorsal commissural nucleus. A loose network of longitudinally or transversely oriented SPN dendrites was located within the dorsal commissural nucleus itself. The lateral margin of the dorsal commissural nucleus was roughly demarcated by longitudinally oriented dendrites. Together with the dendrites of the SPNs of the intercalated and intermediolateral cell column, laterally oriented dendrites of the dorsal commissural nucleus converged and formed the transverse dendritic bundles in the intermediate zone that connect the dorsal commissural nucleus and the intermediolateral cell column. The transverse dendritic bundles were arranged periodically. The axons of the SPNs in the dorsal commissural nucleus traveled laterally into the transverse dendritic bundles, then turned ventrally near the intermediolateral cell column, and finally entered the ventral funiculus. After rhizotomy of the ventral roots of the upper lumbar cord, labeled SPNs were found only on the side contralateral to the rhizotomy. The dorsal commissural nucleus appears as a compact single cell column, but our results clearly show that this nucleus actually consists of two adjacent parallel columns of cells.     

Correlation between adjuvanticity and immunogenicity of cholera toxin B subunit in orally immunised young chickens

The aim of the present study was to investigate whether the adjuvanticity of the cholera toxin B (CTB) subunit was correlated with its immunogenicity in young orally immunised chickens. Thirteen 15-day-old chickens were orally immunised with bovine serum albumin (BSA) glutaraldehyde coupled to CTB. The chicken antibody (IgG) concentrations against BSA and CTB, respectively, were quantified by ELISA. A significant positive correlation (r=0.66, n=39, p<0.001) between the concentrations of immunospecific antibodies with specificities against BSA and CTB, respectively, demonstrated that the adjuvanticity of CTB is correlated with its immunogenicity.     

Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination

The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (non-cholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNA-polymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coli enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 micrograms/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a near-native conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenicity in Peruvian volunteers of a booster dose of oral cholera vaccine consisting of whole cells plus recombinant B subunit.

Forty-nine subjects received two doses of oral cholera vaccine consisting of whole cells plus recombinant B subunit; this was followed by a booster dose one year later. After the primary series, a significant (greater than twofold) increase in the levels of vibriocidal, anti-cholera toxin immunoglobulin G and anti-cholera toxin immunoglobulin A antibodies occurred in 54, 88, and 81% of the subjects, respectively. Within one year, titers decreased to levels close to baseline. A booster dose then induced rises to those which occurred after the initial vaccination. The results suggest that 1-year booster doses may be necessary to maintain immunity against cholera in Latin America.     

一种新的编码霍乱毒素的丝状噬菌体CTAKΦ

目的 探讨介导霍乱弧菌毒素基因水平转移的一种独特的遗传结构。方法 从O139群霍乱弧菌菌株FJ97129上清中分离出丝状噬菌体颗粒CTAKΦ，并进行电镜观察。从丝状噬菌体颗粒CTAKΦ中纯化出DNA，并进行链型分析。对pCTAK进行酶谱分析。用ctxAB、zot引物对pCTAK进行PCR扩增检测，用RS1探针对pCTAK进行Southern blot检测；用pCTAK转化DH5α和IEM101得到DX29和4329，将pCTAK克隆于pUC18载体并转入DH5α得到UD29，用GM1－ELISA检测DX29、4329、UD29的CT表达。对pCTAK的ctxAB、zot基因片段进行测序，并用DNASTAR软件和BLAST算法，在国际互联网上对测序结果进行序列分析。结果 发现和分离了一种编码霍乱毒素的质粒pCTAK，它以稳定的高拷贝数存在于1株天然的O139霍乱弧菌FJ97129中；酶谱分析发现其明显不同于CTX元件酶谱，在CTX元件中相当保守的酶切位点：BglⅡ、EcoRⅤ、PstⅠ、EcoRⅠ，在pCTAK中没有切点。ctxAB、zot引物对pCTAK的PCR扩增检测呈阳性，RS1探针杂交呈阳性；pCTAK的ctxAB、zot基因序列与已报道的序列有很高的同源性。pCTAK能直接或经载体转化DH5α和IEM101并表达CT。从FJ97129培养上清中分离出丝状噬菌体颗粒CTAKΦ，电镜观察并计算其直径大小为7nm左右。其全基因组大小为8.5kb，为单链DNA。结论 发现了一种新的编码霍乱毒素的丝状噬菌体CTAKΦ。     

Safety and Immunogenicity Assessment of an Oral Cholera Vaccine through Phase I Clinical Trial in Korea

The safety, tolerability and immunogenicity of an oral cholera vaccine (OCV) was assessed in adult Korean male through an open-label, non-comparative clinical study. Two doses of vaccine with an interval of 2 weeks were given to 20 healthy subjects. A total of 7 adverse events occurred in 6 subjects. However, no clinically significant change was observed in electrocardiograms, vital signs, physical examinations, and clinical laboratory tests. The immunogenicity of OCV was evaluated by serum vibriocidal assay where anti-Vibrio cholerae O1 and O139 antibodies were measured at day 0, 14, and 28 of vaccine administration. The antibody titers ranged from < 2.5-5,120 for V. cholerae O1 Inaba, < 2.5-10,240 for V. cholerae O1 Ogawa and < 2.5-480 for V. cholerae O139. In addition, the fold increase in antibody titers ranged from 1-4,096 for O1 Inaba, 1-8,192 for O1 Ogawa, and 1-384 for O139. The seroconversion rate was 95% and 45% for O1 and O139 antibodies, respectively. Our study clearly shows that administration of two doses of OCV at a 2 week-interval increases an appropriate level of antibody titer in the serum of healthy Korean adult males (Clinical Trial Number, NCT01707537).

Graphical Abstract