恶性疟原虫裂殖子表面蛋白1羧基端编码基因真核表？…

构建以恶性疟原虫红内期重要的疫苗候选抗原－裂殖子表面蛋白羧基端编码分子量４２０００蛋白的基因片段为外源基因的可用作候选核酸疫苗的真核表达载体。方法目前对疟疾核酸疫苗的研究仅见于鼠疟，将恶性疟原虫ＦＵＰ株裂殖子表面蛋白１羧基端编码４２０００蛋白的基因片段用常规分子克隆方法，分别克隆入非分泌性真核表达载体ＶＲ１０１２和改建后的分泌型载体ＶＲ１０１２／ＴＡ中，通过ＰＣＲ和酶切鉴定出重组克隆。     

恶性疟原虫海南株裂殖子表面蛋白MSP117区基因的测序及其核酸疫苗的构建

恶性疟原虫海南株裂殖子表面蛋白ＭＳＰ１１７区基因的测序及其核酸疫苗的构建①①本研究由联合国开发计划署／世界银行／世界卫生组织热带病研究和培训特别规划署（ＴＤＲ）资助ＩＤ．ＮＯ．９５０３８５作者简介：王晓晨，男，３１岁，助教，硕士；薛采芳，女，５８岁，...     

THE POTENTIAL USES OF GENOTYPING OF PLASMODIUMFALCIPARUMISOLATESBYTHENESTEDPOLYMERASECHAINREACTION

巢式ＰＣＲ方法被应用于泰国疟区（Ｂｏｒａｉ）恶性疟原虫的基因分型。应用特异的等位基因引物扩增了恶性疟原虫裂殖子表面蛋白１（ＭＳＰ１）基因的１、４变异多态区。以凝胶电泳分析扩增的目的基因片段。结果表明：共分别检测出恶性疟原虫６个ＭＡＤ２０等位基因型、４个Ｋ１和１个ＲＯ３３等位基因型。在９个月的流行季节，上述虫株的等位基因频率没有明显的改变，关存在较高程度的恶性疟原虫不同等位基因株的混合感染。本文进一步阐明了巢式ＰＣＲ技术在疟疾流行病学上的应用前景及优点。     

套式PCR扩增特定MSP2基因片段在恶性疟流行病学的应用…

设计３对针对恶性疟原虫裂殖子表面蛋白２（ＭＳＰ２）基因特异的引物，采用套式聚合酶链式反应（ＰＣＲ）技术，扩增ＭＳＰ２基因的中间多态区目的基因，并应用于泰国Ｂｏｒａｉ疟区恶性疟患者血标本分析，结果发现，自然感染的恶性疟原虫株群中，ＭＳＰ２基因存在着多态性，城１５２份恶性疟感染血样中，发现１７种不同的ＭＳＰ２等位基因，其中１２种属于Ｉｎｄｏｃｈｉｎａ（ＩＣ）等位基因家族，５种属于ＦＣ２７等位基因家族，     

恶性疟复合多价抗原基因在小鼠中免疫应答的初步研究

目的探索恶性疟复合多价ＤＮＡ疫苗的可行性。方法把带有ＡＴＧ的接头与人工合成的恶性疟原虫复合多价抗原基因ＡＢ相连后，构建分别带有ＳＶ４０或ＲＳＶ启动子的真核表达载体ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ，重组表达质粒经肌肉注射免疫ＢＡＬＢ／ｃ小鼠后，检测其诱发特异性体液和细胞免疫应答水平及毒副作用。结果ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ免疫ＢＡＬＢ／ｃ小鼠后均诱发了一定水平的细胞及体液免疫应答，带ＲＳＶ启动子的ｐＲＥＰ９／ＡＢ免疫原性略强于带ＳＶ４０启动子的ｐＳＶ２／ＡＢ，ＤＮＡ免疫后未见明显的毒副作用。结论恶性疟复合多价ＤＮＡ疫苗可诱发特异的免疫应答，为疟疾ＤＮＡ疫苗的研究提供了一定的理论及实验依据     

恶性疟原虫保护性抗的复合基因——豆苗病毒重组活疫苗株的构建

将本实验室合成的一段编码恶性疟原虫不同发育阶段抗原表位基因，包括裂殖子表面怕ＭＳＡ１、ＭＳＡ２、环子孢子蛋白ＣＳＰ及环状体感染经细胞表面蛋白ＲＥＳＡ以及来自白细胞介素－１（ＩＬ－１）和破伤风类毒素（ＴＴ）上Ｔ细胞激活点等的抗原基因（ＨＧＦＳＰ）。先定向克隆人ＰＳＫ载体的ＥｃｏＲＩ、ＢａｍＨＩ位点，后用ＥｃｏＲＩ单酶切，补平及用ＳａｃⅠ单酶切下手目的基因再定向克隆人痘苗病毒表面载体ＰＪ２－１６的Ｓｍ     

恶性疟复合多价抗原基因在小鼠中免疫应答的初步 …

目的 探索恶性疟复合多价ＤＮＡ疫苗的可行性。方法 把带有ＡＴＧ的接头与人工合成的恶性疟原虫复合多价抗原基因ＡＢ相连后，构建分别带有ＳＶ４０或ＲＳＶ启动子的真核表达载体ｐＳＶ／２ＡＢ及ｐＲＥＰ９／ＡＢ，重组表达质粒经肌肉注射免疫ＢＡＬＢ／ｃ小鼠后，检测其诱发特异性体液和细胞免疫应答水平及毒副作用。结果 ｐＳＶ２／ＡＢ及ｐＲＥＰ９／ＡＢ免疫ＢＡＬＢ／ｃ小鼠后均诱发了一定水平的细胞及体液免疫应答，带ＲＳ     

巢式PCR在恶性疟原虫基因分型上的应用研究

巢式ＰＣＲ方法被应用于泰国疟区恶性疟原虫的基因分型，应用特异的等位基因引物扩增了恶性疟原虫裂殖子表面蛋白基因的１、４变异多态区，以凝胶电泳分析扩增的目的基因片段，结果表明；共分别检测出恶性疟原虫６个ＭＡＤ２０等位基因型，４个Ｋ和１个Ｒ０３３等位基因型。在９个月的流行季节，上述虫株的等位基因频率没有明显的改变，并存在较高程度的恶性疟原虫不同等位基因株的混合感染，本文进一步阐明了巢式ＰＣＲ技术在疟疾流     

套式PCR扩增特定MSP2基因片段在恶性疟流行病学上的应用研究

设计３对针对恶性疟原虫裂殖子表面蛋白２（ＭＳＰ２）基因特异的引物，采用套式聚合酶链式反应（ＰＣＲ）技术，扩增ＭＳＰ２基因的中间多态区目的基因，并应用于泰国Ｂｏｒａｉ疟区恶性疟患者血标本分析。结果表明：自然感染的恶性疟原虫株群中，ＭＳＰ２基因存在着多态性。在１５２份恶性疟感染血样中，发现１７种不同的ＭＳＰ２等位基因。其中１２种属于Ｉｎｄｏｃｈｉｎａ（ＩＣ）等位基因家族，５种属于ＦＣ２７等位基因家族。ＩＣ等位基因型较为常见。在９个月的流行季节，ＩＣ和ＦＣ２７等位基因型的频率保持相对稳定。较高程度的两种等位基因型混合感染的现象也被发现。因此提示，套式ＰＣＲ方法不仅提高了疟疾诊断水平，更重要的是，为疟疾的流行病学研究开辟了新的途径     

恶性疟原虫疫苗研究VⅡ.PfCMR基因与人白细胞介素—2基因…

用限制性内切酶ＥｃｏＲＩ和Ｓａｌｌ将恶性疟原虫复合抗原基因ＰｆＣＭＲ从质粒ＰＷＲ４５０－１／ＰｆＣＭＲ中切下，插入质粒ＰＢＶ２２０／ＩＬ－２中人白细胞介素－２（ＩＬ－２）基因的ＥｃｏＲＩ位点，重组质粒转化大肠杆菌ＤＨ５ａ，通过ＰＣＲ扩增和酶切鉴定，筛选出自向插入的重组载体ＰＢＶ２２０／ＰｆＣＭＲ－ＩＬ－２。为表达ＰｆＣＭＲ－ＩＬ－２融合蛋白打下基础。     

携带幽门螺杆菌ureB和IL-2质粒DNA的减毒鼠伤寒沙门菌疫苗构建

目的　构建编码幽门螺杆菌 (Helicobacterpylori,H .pylori)尿素酶B亚单位 (ureB)基因和小鼠IL 2基因以减毒鼠伤寒沙门菌为载体的核酸疫苗 ,体外转染Cos 7细胞 ,鉴定其表达蛋白的免疫性。方法　应用聚合酶链式反应 (PCR)技术从H .pylori标准菌株CCUG1 7874基因组DNA扩增ureB基因 ,从重组质粒 pCIneo IL 2扩增小鼠IL 2基因 ,通过T A克隆分别插入 pUCmT载体 ,检测ureB及IL 2的核苷酸序列 ,通过酶切、连接反应分别克隆入真核表达载体 pIRES ,PCR和酶切反应进行鉴定 ;重组载体 pIRES ureB和 pIRES ureB IL 2转入减毒鼠伤寒沙门菌LB50 0 0 ,抽提质粒 ,再次转入SL72 0 7,反复传代 ,鉴定核酸疫苗载体菌的稳定性。通过脂质体法将重组载体 pIRES ureB和pIRES ureB IL 2转染Cos 7细胞 ,SDS PAGE及Westernblot法检测表达蛋白的免疫性。结果　扩增出长约 1 70 0bp的ureB基因和 51 0bpIL 2 ,测序结果表明 ,扩增出的ureB基因与基因库H .pyloriureB序列一致 ,IL 2序列和小鼠的IL 2序列一致 ,PCR和酶切鉴定结果证实ureB和IL 2基因克隆入真核表达载体 pIRES ,并成功构建了稳定的幽门螺杆菌ureB和IL 2基因以减毒鼠伤寒沙门菌为载体的核酸疫苗 ,并且以Westernblot检测到特异性的相对分子质量 (Mr)为 6 6× 1 0 3的UreB蛋白     

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.     

Protective immune responses to the 42-kilodalton (kDa) region of Plasmodium yoelii merozoite surface protein 1 are induced by the C-terminal 19-kDa region but not by the adjacent 33-kDa region.

Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP1(42)) or its 19-kDa C-terminal processing product (MSP1(19)) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP1(33)) of MSP1(42) also induces protection, the gene segment encoding MSP1(33) was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP1(33) and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP1(33) failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP1(19) were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP1(33) had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP1(19). The finding that P. yoelii MSP1(33) elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP1(42).     

Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1

Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1(42)) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP1(42) was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1(42) and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1(42). However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP1(19)). Vaccination of nonhuman primates with E. coli MSP1(42) in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1(42), is a viable candidate for human vaccine trials.     

Immunogenicity and protective efficacy of a Plasmodium yoelii Hsp60 DNA vaccine in BALB/c mice

The gene encoding the 60-kDa heat shock protein of Plasmodium yoelii (PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 microg of PyHsp60 DNA vaccine alone or in combination with 30 microg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognized P. yoelii sporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50 P. yoelii sporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test; P = 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 x 10(3) irradiated P. yoelii sporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation.     

Immunity to recombinant plasmodium falciparum merozoite surface protein 1 (MSP1): protection in Aotus nancymai monkeys strongly correlates with anti-MSP1 antibody titer and in vitro parasite-inhibitory activity

A number of malarial blood-stage candidate vaccines are currently being tested in human clinical trials, but our understanding of the relationship between clinical immunity and data obtained from in vitro assays remains inadequate. An in vitro assay which could reliably predict protective immunity in vivo would facilitate vaccine development. Merozoite surface protein1 (MSP1) is a leading blood-stage malaria vaccine candidate, and anti-MSP1 antibodies from individuals that are clinically immune to malaria inhibit the invasion of Plasmodium merozoites into erythrocytes in vitro. Using expression in Escherichia coli and subsequent refolding, we have produced two allelic forms of MSP1(42) (FVO and 3D7). Aotus nancymai monkeys were immunized with MSP1(42)-FVO, MSP1(42)-3D7, or a combination of FVO and 3D7 allelic forms, (MSP1(42)-C1) and were subsequently challenged with Plasmodium falciparum FVO parasites. Sera obtained prior to challenge were tested by standardized enzyme-linked immunosorbent assay (ELISA) to determine antibody titer, and immunoglobulin G (IgG) fractions were also obtained from the same sera; the IgG fractions were tested in an in vitro growth inhibition (GI) assay to evaluate biological activity of the antibodies. Regardless of the immunogen used, all monkeys that had >200,000 ELISA units against MSP1(42)-FVO antigen before challenge controlled their infections. By contrast, all monkeys whose purified IgGs gave <60% inhibition activity in an in vitro GI assay with P. falciparum FVO required treatment for high parasitemia after challenge. There is a strong correlation between ELISA units (Spearman rank correlation of greater than 0.75) or GI activity (Spearman rank correlation of greater than 0.70) and protective immunity judged by various parameters (e.g., cumulative parasitemia or day of patency). These data indicate that, in this monkey model, the ELISA and GI assay values can significantly predict protective immunity induced by a blood-stage vaccine, and they support the use of these assays as part of evaluation of human clinical trials of MSP1-based vaccines.     

Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial

In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system. One recombinant vaccine, bvMSP1(42), based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P. falciparum. The second recombinant protein, P30P2MSP1(19), has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed in Saccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP1(42), for efficacy in the A. nancymai system. The new formulation of P30P3MSP1(19) performed significantly worse than bvMSP1(42) and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.     

hLKB1基因重组质粒构建及蛋白表达和定位

目的构建hLKB1真核表达载体,证实融合蛋白在胃癌细胞内的表达及定位。方法提取胃正常黏膜上皮细胞GES-1的总mRNA并进行反转录。以反转录的cDNA为模板PCR扩增hLKB1全长编码基因,克隆至pCDNA3.1/flag的真核表达载体中。将构建的重组质粒测序并转染到胃癌SGC-7901细胞中,分别利用Western blot和激光共聚焦扫描显微技术检测其表达和在胃癌细胞中的定位。结果 hLKB1全长基因序列克隆到真核表达载体中,酶切鉴定片段为1300bp。Western blot检测到真核转染的Flag-hLKB1在胃癌SGC-7901细胞表达,条带约为50kD,免疫荧光显示蛋白广泛定位于细胞质和细胞核内。结论成功构建了hLKB1基因真核表达载体,并验证其在胃癌细胞表达。     

B 群脑膜炎球菌外膜蛋白0315 不同形式疫苗免疫效果比较

目的:初步探讨和评价NMB0315 核酸疫苗、重组蛋白疫苗及核酸疫苗+重组蛋白疫苗联合免疫诱导小鼠产生的特异性体液/ 细胞免疫应答水平及其免疫保护效果,为进一步探索NMB0315 疫苗有效的免疫方法和途径提供实验依据。方法:大量制备核酸疫苗[pcDNA3.1(+) / NMB0315]和重组蛋白疫苗(pET-30a/ NMB0315),采用核酸初免-蛋白加强的方法联合免疫或分别免疫雌性BALB/ c 小鼠,测定特异性体液/ 细胞免疫应答水平、免疫血清体外杀菌效价,观察疫苗对感染B 群脑膜炎球菌小鼠的免疫保护效果。结果:NMB0315 核酸疫苗组(pNMB0315-CpG)、蛋白疫苗组(rNMB0315-FA)及联合免疫疫苗组(pNMB0315-CpG+rNMB0315-FA)诱导的血清特异性IgG、IgG1、IgG2a 及生殖道灌洗液中特异性sIgA 水平在第八周达到峰值,A450 值分别为(0.505±0.042、0.513±0.022、0.342±0.017、0.250±0.015)、(0.823±0.061、0.807±0.045、0.596±0.027、0.450±0.028)和(0.694±0.053、0.711±0.032、0.455±0.021、0.386±0.024),明显高于PBS 对照组(P<0.01);其中,蛋白疫苗组的抗体水平明显高于联合免疫疫苗组和核酸疫苗组(P<0.05)。小鼠脾淋巴细胞刺激指数及IFN-γ酌水平,联合免疫疫苗组明显高于蛋白疫苗组和核酸疫苗组(P<0.05);核酸疫苗组、蛋白疫苗组和联合免疫疫苗组免疫血清在补体介导下的体外杀菌抗体效价分别为1 :64、1 :128 和1 ：128,对实验小鼠的免疫保护率分别为70%、95%和80%。免疫2、4、6、8 周时,核酸疫苗组、重组蛋白疫苗组和联合免疫疫苗组IgG2a/ IgG1 比值均小于1。结论:NMB0315 疫苗诱导的体液免疫(包括黏膜免疫)效果从高到低为:重组蛋白疫苗组、联合免疫疫苗组、核酸疫苗组;诱导细胞免疫的效果从高到低为:联合免疫疫苗组、核酸疫苗组、重组蛋白疫苗组; NMB0315 疫苗对实验小鼠的免疫保护效果从高到低为:重组蛋白疫苗组、联合免疫疫苗组、核酸疫苗组。