恶性疟原虫裂殖子嵌合性表面蛋白疫苗诱导其高滴度生长抑制抗体

<正>恶性疟原虫裂殖子表面蛋白1的C端19 000区(Pf MSP119)是保护性抗体的标靶,但是,一个主要的血期候选疫苗pf MSP142其中包含了Pf MSP119的保护性表位,临床试验表明它的免疫原性和效力不是最佳的。根据用约氏疟原虫小鼠模型所作的概念验证研究,作者将重组的Pf MSP119(rPf MSP119)与恶性疟原虫裂殖子缺失了其低复杂性的     

恶性疟裂殖子表面蛋白1合成基因在毕赤酵母中的表达

恶性疟原虫裂殖子表面蛋白1是当今疟疾疫苗主要的候选抗原。由于天然MSP1基因AT含量异常高(为74%),使得克隆全长天然基因无法实现。本文已全合成了msp1基因(4940bp),解决了该天然基因在异源系统中不稳定的问题。为制备大量msp1重组蛋白进行疫苗有效性试验,本研究建立了msp1基因在毕赤酵母中的表达,将合成的msp1基因克隆到毕赤酵母胞内表达载体pPIC3.5,构建了重组质粒pPIC3.5/msp1,用电击转化毕赤酵母得到重组转化子,经PCR证实msp1基因已整合于毕赤酵母染色体中。含有重组表达质粒的毕赤酵母菌经甲醇诱导后表达出全长msp1重组蛋白。表达产物能与识别MSP1分子二硫键依赖构象表位的特异性单抗发生很强的反应,表明msp1重组蛋白至少在该表位构象上与天然蛋白一致。从毕赤酵母中分离得到大量msp1为开展该蛋白的结构与功能,特别是测定其疟疾保护性免疫提供可能。     

重组恶性疟原虫DNA质粒免疫小鼠制备单克隆抗体

用恶性疟原虫MSP131基因片段的重组质粒DNA直接免疫BALB/c小鼠,诱导产生体液,免疫后取脾细胞与SP2/0小鼠骨髓瘤细胞在PEG1450作用下进行融合,获得了2株能分泌抗恶性疟原虫MSP131单克隆抗体的小鼠杂交瘤细胞株9H9和8A2。用酶联免疫吸附试验检测,小鼠腹水抗体滴度最高为1∶10 000。经免疫球蛋白类型和亚类鉴定,2株杂交瘤细胞株均为IgG\-1\.蛋白免疫印迹试验表明,此单克隆抗体与MSP1\|31蛋白抗原有特异免疫反应,证明通过质粒DNA直接免疫小鼠可制备特异性单克隆抗体。     

以ISA720为佐剂甘氨酸作为保护剂的恶性疟疾疫苗AMA1的长期稳定性

<正>恶性疟原虫顶端膜抗原(AMA1)是恶性疟原虫无性繁殖血液期表达的蛋白,是恶性疟疾疫苗的候选抗原。AMA1蛋白是疟原虫FVO和3D7等位基因表达产物,恶性疟疾候选疫苗AMA1-C1/ISA720     

以ISA720为佐剂甘氨酸作为保护剂的恶性疟疾疫苗AMAl的长期稳定性

恶性疟原虫顶端膜抗原（AMA1）是恶性疟原虫无性繁殖血液期表达的蛋白,是恶性疟疾疫苗的候选抗原。AMA1蛋白是疟原虫FVO和3D7等位基因表达产物,恶性疟疾候选疫苗AMA1-C1／ISA720是AMA1抗原与佐剂MontanideISA720按照1：1（质量比）配伍混合合成的。为了将来在大规模人群中接种的需要,     

新型Aβ42寡聚体模拟表位疫苗的制备

旨在获得一株特异靶向致病性Aβ42寡聚体的模拟表位疫苗。在前期工作中,应用亲和层析的方法从人静脉用免疫球蛋白(Intravenous immune globulin,IVIG)中纯化获得特异性识别Aβ42寡聚体的抗体组分IVIG-AOB。以IVIG-AOB为靶蛋白,应用噬菌体展示技术淘选出一系列Aβ42寡聚体的特异性环状模拟表位多肽,随后将表位多肽融合表达至乙肝病毒核心蛋白(HBcVLP)构建成表位载体疫苗,进而将免疫小鼠,从中筛选出能有效免疫产生抗Aβ42寡聚体抗体的模拟表位疫苗。从噬菌体环形七肽库中筛选出5条特异性结合IVIG-AOB的模拟表位多肽,将其克隆至HBc载体,并在大肠杆菌中成功表达和组装成嵌合表位的HBc-VLP,通过免疫小鼠优选出一株效果最好的Aβ42寡聚体构象表位疫苗HBc-C4。Aβ寡聚体是AD发生发展的主要致病因素。基于Aβ42寡聚体独特的构象表位研制的表位HBc-VLP疫苗诱导机体产生的抗体将能够特异靶向并中和毒性Aβ42寡聚体,而不影响Aβ42的正常生理功能,从而起到从根本上治疗AD的作用。     

肝素/硫化肝素在恶性疟原虫入侵红细胞过程中的作用机制

疟疾是疟原虫通过雌性按蚊为媒介传播的寄生虫病,是当今世界公共卫生的突出问题.寄生于人体的疟原虫主要包括恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫和诺氏疟原虫5种,其中,恶性疟原虫的致病性最为强烈,是导致全球疟疾发病和死亡的重要病原体.硫化肝素是广泛分布于脊椎动物细胞表面的无支链多糖,为疟原虫入侵宿主红细胞的一个重要受体,在疟原虫入侵过程中发挥着重要的作用.疟原虫入侵宿主红细胞是一个快速而复杂的过程,恶性疟原虫入侵相关蛋白质在疟原虫入侵过程中具有重要的作用,主要参与黏附宿主红细胞、形成移动连接复合体、形成和修饰纳虫空泡等过程.黏附是恶性疟原虫入侵宿主红细胞的第一步,入侵相关蛋白质与宿主红细胞表面硫化肝素受体结合,继而完成后续的入侵.综合国内外文献,本文围绕肝素/硫化肝素在治疗恶性疟疾中的应用、与恶性疟原虫入侵相关蛋白质的结合以及恶性疟原虫入侵相关蛋白质在裂殖子入侵宿主红细胞过程中的作用机制等研究方面进行综述,为恶性疟原虫入侵过程中相关蛋白质与肝素结合的分子机制提供理论依据,同时也为新型抗疟药物和疫苗的研制以及治疗提供参考.     

N端9个氨基酸缺失对恶性疟融合抗原免疫原性的影响

由两个疟疾疫苗候选抗原AMA 1(III)和MSP1 19融合而成的恶性疟原虫融合抗原 2 (PfCP 2 ) ,是一个很有应用前景的疟疾疫苗候选抗原。但PfCP 2表达产物为双带型 ,这影响到该疫苗候选抗原作为产品进入临床试验。为此分析了表达产物N末端的氨基酸序列 ,发现其中低分子量条带的N末端不完整 ,缺失 9个氨基酸。进一步用PCR技术对PfCP 2进行改建 ,使其N末端缺少 9个氨基酸 ,产生PfCP 2 .9基因。实验结果显示 :PfCP 2 .9在毕赤酵母中的表达产物为单一条带 ,且在表达水平、二硫键依赖的构象、免疫原性及免疫血清抑制疟原虫生长等方面与PfCP 2一致。PfCP 2表达产物双带型问题得到解决 ,为该融合抗原疟疾疫苗进入临床试验排除了重要障碍。     

恶性疟原虫CSP基因在毕赤酵母中的分泌表达

目的用毕赤酵母表达系统表达恶性疟原虫环子孢子蛋白(CSP),便于恶性疟疾疫苗的进一步研究。方法根据Gen Bank得到恶性疟原虫7G8的基因序列,选取该序列628~1 194位基因,以密码子最佳化为原则合成基因后构建酵母表达载体CSP/p GAPZa A,电转毕赤酵母菌PDI-GS115,获得酵母重组体。三角瓶规模表达重组菌,获得表达上清。结果经SDS-PAGE电泳、Western blot检测表达上清,均显示有目的蛋白表达。结论在毕赤酵母中实现了CSP基因的表达,为恶性疟疾疫苗的研发作了必要的补充。     

人乳头瘤病毒L1基因与丹毒丝菌SpaA-N优势抗原表位序列的嵌合重组质粒构建及活性鉴定

为确定丹毒丝菌表面保护性抗原A的N-末端(SpaA-N)优势抗原表位,研发新型表位DNA嵌合疫苗,利用生物信息学软件对丹毒丝菌SpaA-N的优势B细胞抗原表位进行预测,以重叠PCR将优势表位插入人乳头瘤病毒16型的主要衣壳蛋白( HPV-16 LI) HI环结构的编码序列中,构建获得重组嵌合质粒pcDNA3-HPV-LI -△spaA.该重组质粒经体内、外瞬时特染后,RT-PCR均扩增获得了1 500 bp左右的目的片段.免疫印迹试验显示,体外转染嵌合质粒的细胞总蛋白能够与GST-SpaA-N重组蛋白免疫血清在56 kD处产生特异性结合.ELISA分析显示嵌合质粒可在小鼠体内产生差异显著的特异性抗体(P＜0.01),抗体滴度为1:1 000.此外,pcDNA3-HPV-L1-△spaA制备的抗血清至少可在1∶10稀释度条件下,介导外源补体对半数以上的菌体进行杀伤.该研究表明,获得的SpaA-N表位DNA嵌合疫苗具有免疫活性,为研发安全有效的丹毒丝菌新型DNA疫苗提供了一个新思路.     

High-Level Production and Purification of P30P2MSP119, an Important Vaccine Antigen for Malaria, Expressed in the Methylotropic Yeast Pichia pastoris

P30P2MSP1(19) is a recombinant subunit vaccine derived from merozoite surface protein 1 (MSP1) of Plasmodium falciparum, the causative agent of malaria. P30P2MSP1(19) consists of two universal T-cell epitopes fused to the most C-terminal 19-kDa portion of MSP1, and this protein has previously shown promising potential as a vaccine for malaria. However, previous attempts at producing this molecule in Saccharomyces cerevisiae resulted in the production of a truncated form of the molecule missing most of the universal T-cell epitopes. Here, we report the production of full-length P30P2MSP1(19) in Pichia pastoris. As salt precipitation is a common problem during P. pastoris high-density fermentation, we utilized an alternative low-salt, fully defined medium that did not reduce growth rates or biomass yields to avoid precipitation. A total of 500 mg/L of secreted purified protein was produced in high cell density fermentation and the protein was purified in one step utilizing nickel-chelate chromatography. P30P2MSP1(19) produced in Pichia was reactive with monoclonal antibodies that recognize only conformational epitopes on correctly folded MSP1. Rabbits immunized with this molecule generated higher and more uniform antibody titers than rabbits immunized with the protein produced in Saccharomyces. P30P2MSP1(19) produced in Pichia may prove to be a more efficacious vaccine than that produced in Saccharomyces and Pichia would provide a system for the cost-effective production of such a vaccine.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

High-level expression and immunogenic properties of the recombinant rabbit hemorrhagic disease virus VP60 capsid protein obtained in Pichia pastoris

The VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) (Spanish isolate AST/89) was cloned and expressed in Pichia pastoris. The transformed yeast was grown at high cell density and an expression level of about 1.5 g VP60L(-1) culture was obtained. The protein was detected associated with the cell debris fraction of the recombinant yeast after mechanical disruption. It was purified by a simple method and was obtained N-glycosylated with purity of approximately 70% as deduced from densitometry scan analysis. The recombinant product was antigenically similar to the native capsid protein as determined with polyclonal antibodies obtained from rabbits vaccinated with VP60 protein purified from native virus. The immunogenicity of VP60 protein purified from P. pastoris was demonstrated by ELISA in a vaccination experiment conducted with two groups of rabbits subcutaneously immunized. Animals vaccinated with VP60 in Freund's incomplete adjuvant developed a significant (p<0.01) virus-specific antibody response while the group injected with placebo remained seronegative. Preliminary results showed that the antigen administered within the cell debris fraction of the transformed yeast protected rabbits immunized by the oral route against an intramuscular challenge with 100 LD50 (16,000 hemagglutination units) of homologous virus.     

Fusion of two malaria vaccine candidate antigens enhances product yield, immunogenicity, and antibody-mediated inhibition of parasite growth in vitro

A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and approximately 1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1-19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1-19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.     

Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development

The newly identified GPI-anchored Plasmodium vivax merozoite surface protein 1 paralog (MSP1P) has a highly antigenic C-terminus that binds erythrocytes. To characterize the antigenicity and immunogenicity of two regions (PvMSP1P-19 and -33) of the highly conserved C-terminus of MSP1P relative to PvMSP1-19, 30 P. vivax malaria-infected patients and two groups of mice (immunized with PvMSP1P-19 or -33) were tested for IgG subclass antibodies against PvMSP1P-19 and -33 antigens. In the patients infected with P. vivax, IgG1 and IgG3 levels were significantly higher than those levels in healthy individuals, and were the predominant response to the two C-terminal fragments of PvMSP1P (p < 0.05). In mice immunized with PvMSP1P-19, IgG1 levels were the highest while IgG2b levels were similar to IgG1 levels. The levels of Th1 cytokines in mice immunized with PvMSP1P-19 or -33 were significantly higher than those in mice immunized with PvMSP1-19 (p < 0.05). Our results indicate that: (i) IgG1 and IgG3 (IgG2b in mice) are predominant IgG subclasses in both patients infected with P. vivax and mice immunized with PvMSP1P-19 or -33; (ii) the C-terminus of MSP1P induces a Th1-cytokine response. This immune profiling study provides evidence that MSP1P may be a potential candidate for vivax vaccine.     

FMDV VP1基因与HSP70基因在毕赤酵母中的融合表达及免疫特性

利用毕赤酵母系统对O型口蹄疫病毒VP1基因与结核杆菌HSP70基因进行融合表达,并检测此融合蛋白对小鼠细胞免疫和体液免疫的影响。将人工合成的O型口蹄疫病毒VP1基因与结核杆菌HSP70基因克隆入酵母表达载体pPICZαA中,以电穿孔法转化酵母菌X-33,用Zeocin YPDS平板筛选重组子,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物。以皮下接种的方式给小鼠进行3次免疫,同时设两组对照,分别免疫PBS和常规灭活疫苗,然后通过MTT法和ELISA分别检测淋巴细胞的增殖情况和抗体水平。结果表明融合蛋白既能诱导细胞免疫应答又能诱导体液免疫应答,其诱导产生的抗体水平略低于常规灭活疫苗,而细胞免疫水平则高于后者。     

Interaction of malaria parasite-inhibitory antibodies with the merozoite surface protein MSP1(19) by computational docking

Merozoite surface protein 1 (MSP1) of the malaria parasite Plasmodium falciparum is an important vaccine candidate antigen. Antibodies specific for the C-terminal maturation product, MSP1(19), have been shown to inhibit erythrocyte invasion and parasite growth. Specific monoclonal antibodies react with conformational epitopes contained within the two EGF-like domains that constitute the antigen MSP1(19). To gain greater insight into the inhibitory process, the authors selected two strongly inhibitory antibodies (designated 12.8 and 12.10) and modeled their structures by homology. Computational docking was used to generate antigen-antibody complexes and a selection filter based on NMR data was applied to obtain plausible models. Molecular Dynamics simulations of the selected complexes were performed to evaluate the role of specific side chains in the binding. Favorable complexes were obtained that complement the NMR data in defining specific binding sites. These models can provide valuable guidelines for future experimental work that is devoted to the understanding of the action mechanism of invasion-inhibitory antibodies.     

兔出血症病毒衣壳蛋白基因在毕赤酵母中的表达

将RHDVVP60基因插入酵母转移载体pPICZB中转化毕赤酵母菌GS115株 ,经筛选获得染色体基因组中整合入VP60基因的重组酵母菌。以甲醇诱导培养后经SDS-PAGE和Westernblot检测表达产物 ,在60kD处出现一特异蛋白条带 ,表明RHDV的衣壳蛋白得到了成功表达。血凝试验表明 ,表达的重组蛋白具有血凝特性 ,可以凝集人“O”型红细胞 ,血凝价达 2.8,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组酵母表达的衣壳蛋白可以在酵母菌体内自聚成大小约4.0nm ,和天然RHDV病毒子在物理形态上类似的病毒样颗粒 (VLPs)。该病毒样颗粒与兔抗RHDV高免血清作用后可被凝集成团 ,表明该VLPs与天然RHDV在抗原性上也极为相似。     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。