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Immunogenicity of recombinant attenuated Salmonella typhimurium expressing a 45-peptide hybrid antigen gene of Plasmodium falciparum 被引量:2
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作者 黄建生 王昌才 +2 位作者 任大明 钟雄林 陈仕荣 《Journal of Medical Colleges of PLA(China)》 CAS 1997年第2期166-171,共6页
A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1,... A synthetic hybrid 45-peptide gene of Plasmodium falciparum (Pf), which encoded two CSP repeated peptides NANP and three merozoite peptides SPf83. 1, SPf55. 1 and SPf35. 1, was cloned in an expression vector pWR450-1, then the recombinant plasmid pWRA was introduced into the attenuated Salmonella typhimurium SL3261. When used as a live vaccine and administered orally (po), intravenously (iv) or intraperitoneally (ip),the recombinant strain was able to live in vivo and elicit specific humoral and cellular immunity in BALB/c mice and rabbits. As oral immunization is safe and effective, it is thought that the live recombinant Salmonella tyPhimurium vaccine may bring the Pf oral live vaccine a step nearer. 展开更多
关键词 Salmonella TYPHIMURIUM plasmodium falciparum hybrid antigen gene oral live vaccine malaria vaccine IMMUNOGENICITY
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Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice 被引量:7
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作者 Yi-Ping Xing Zu-Hu Huang +4 位作者 Shi-Xia Wang Jie Cai Jun Li Te-Hui W Chou Shan Lu 《World Journal of Gastroenterology》 SCIE CAS CSCD 2005年第29期4583-4586,共4页
AIM: To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plas... AIM: To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice. 展开更多
关键词 dna vaccine Hepatitis B virus core antigen IMMUNOGENICITY gene gun CTL HBV
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Subcloning,sequencing and expressing of conserved blocks of P190 gene of Plasmodium falciparum FCC1/HN strain
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作者 杨树桐 潘卫庆 +1 位作者 邓海琳 陆德如 《Journal of Medical Colleges of PLA(China)》 CAS 1993年第3期246-253,共8页
190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according t... 190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according to the sequence of MAD20 strain,with a GCclamp and BamH Ⅰ site at the 5’-end of each one,and a GC clamp and Xba Ⅰ site at the 3’-endof each one.The primers were synthesized by phosphoramidite approach (User’s Manual ofABI Company) and purified using HPLC.Three fragments in the second,third and fourth con-served regions of P190 gene of Plasrnodium falciparum FCC1/HN strain isolated from theblood of patients in Hainan Province of China were amplified by the polymerase chain reaction(PCR).The amplified fragments were suhcloned into pUC18 vectors and sequenced using thedideoxy chain termination method.All three regions of P190 gene of FCC1/HN strain also werehighly conservative as compared with P190 gene of MAD20 (Papua New Guinea isolate),K1(Thailand isolate),Wellcome (West Africa isolate) and CAMP (Malaysia) strains ofPlasmodium falciparum.The C at position 81 in the second conserved block of P190 gene ofFCC1/HN isolate was substituted by T,which did not change the amino acid determined by thecodon corresponding to the substitution.The genes sequenced were cloned into rpGEX-2T,aglutathione S-transferase gene fusion system for expression. 展开更多
关键词 plasmodium falciparum MEROZOITE surface antigen l CONSERVED sequences vaccine
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Subcloning and expression of two conserved regions (Ⅰ,Ⅴ) onP190 antigen of Plasmodium falciparum in E.coli
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作者 潘卫庆 杨树桐 +1 位作者 邓海琳 陆德如 《Journal of Medical Colleges of PLA(China)》 CAS 1994年第1期36-39,共4页
Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction f... Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667)respectively, which encode amino acid residues of conserved region Ⅰ and Ⅴ on the P190 antigen were amplified by polymerase chain reaction from genomic DNA in FCCl/HN strain of Plasmodium falciparum isolated from Hainan Province, China. It was found that there were five base substitutions in the P190CR V, in comparison with the nucleotide sequences of MAD20 strain. These two fragments sequenced were inserted into pGEX-2T plasmid. E. coli JM109 (DE3) were transformed with the recombinant plasmids and the parental plasmid. The results showed that the two fragments were expressed as high-level C-terminal fusions with glutathione S-transferase (GST). The fusion proteins were easily purified from bacterial lysates by affinity chromatography using glutathione Sepharose 4B. 展开更多
关键词 P190 antigen plasmodium falciparum malarial vaccine genetic engineering
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The development of a recombinant hybrid protein of Plasmodium falciparum and analysis of its antigenicity and protectivity
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作者 李全贞 李英杰 +3 位作者 谢毅 任大明 毕惠祥 徐秉锟 《Journal of Medical Colleges of PLA(China)》 CAS 1994年第3期161-169,共9页
A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was ... A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was synthesized chemically. This gene (named HGFC) was cloned and connected with another hybrid gene (HPFA) synthesized previously to make a bigger hybrid gene (HGFCAC). HGFC and HGFCAC were cloned in an expression vector pWR450-1 and transformed into E. Coli JM109. The engineered bacteria could express fusion proteins with molecular weights of 65 and 77 kDa after inducing with isopropylthio-β-D-galactoside (IPTG). The expression rate was about 35% of total bacterial proteins. The expressed products showed sepcific immunological reaction with rabbit antibodies against P. falciparum peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala (EENVEHDA)by Western blotting. The fusion proteins were pruified by precipitation with amonium sulfate. gel filtration and ion-exchange chromatography and the purity was 82%. The purified protein reacted specifically with mouse immune serum against falciparum blood stage antigens by dot enzyme-linked immunosorbent assay (dot-ELISA).The fusion protein was emulsified with Freund's complete adjuvant (FCA) and used to immunize rabbits. The immune serum can recognize P. falciparum erythrocytic stage antigens of Fcc-1/HN strain and Yunnan strain and had weak cross reaction with P. vivax,but had no reaction with P. cynomlogi and P. berghei antigens. The protective effect of the antibody was tested by in vitro inhibition test to cultured falciparum parasites. Preliminary results indicated that the immune sera could effectively reduce the invasion rates of the parasites to red blood cells and inhibit the growth of the in vitro cultured falciparum parasites. The inhibitory capacity of the immune sera to parasite invasion is enhanced as the amount of the sera increases and the incubation time of the sera with the parasites is prolonged.It was shown that after 72 h incubation at 20% concentration with the parasites, the serum can suppress the multiplication of P.falciparum growth in vitro to a level of 80%.The immune sera caused dispersion of the parasite cytoplasm,atrophy of parasites,agglutination of free merozoites and degeneration of schizonts. 展开更多
关键词 plasmodium falciparum RECOMBINANT PROTEIN HYBRID antigen inhibition test in vitro malarial vaccine
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Construction of a vaccinia virus vectored multi--epitope live vaccine candidate for Plasmodium falciparum
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作者 董文其 李明 +1 位作者 毕惠祥 李英杰 《Journal of Medical Colleges of PLA(China)》 CAS 1999年第2期119-123,共5页
To construct live recombinant vaccinia virus, The HGFSP gene encoding CSP, MSA1, MSA2, RESA,IL-1 and TT epitopes was inserted into the Eec RI and Burn HI sites of pSK plasmid. After digested with Eco RI,bluntly ended ... To construct live recombinant vaccinia virus, The HGFSP gene encoding CSP, MSA1, MSA2, RESA,IL-1 and TT epitopes was inserted into the Eec RI and Burn HI sites of pSK plasmid. After digested with Eco RI,bluntly ended by Klenow enzyme and digested with Sac I, the HGFSP gene was cloned into the Sma I and Sac I sites of the vaccinia virus insertion vector (pJ2--16). Recombinant plasmids were identified by gel electrophoresis,restriction enzyme and enzyme map. Results evidenced that HGFSP gene fragment was correctly inserted into the cloning site of hemagglutinin (HA) gene of the pJ2--16 vector. The recombinant plasmids were trans feeted into Cos--7 cells, which were infected with wild type of vaccinia virus Tiantan strain, by means of lipofectamine. Two recombinant vaccinia viruses (HA) were screened and cloned by chicken hemadorption test in BHK21 cells. Indirect immunofluorescence assay (IFA), Dot--ELISA and Western blot with the antibodies against HGFSP protein expressed by E. colt showed that one of the 2 recombinant vaccinia virus expressed desired proteins in infected BHK21 cells. Western blot also showed that the molecular weight of 2 of expressed protein bands was about 23 kDa, according to the theoretical molecular weight of HGFSP protein. Further identification of immunological characters of recombinant virus is under way. 展开更多
关键词 plasmodium falciparum VACCINIA virus gene vaccine
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Analysis of var genes cloned from a Plasmodium falcivarum isolate in China
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作者 Ning Jiang Li Meng +5 位作者 Hui-Jun Lu Wei Kang Shuai Peng Wei-Qing Pan Ji-Gang Yin Qi-Jun Chen 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2012年第2期85-90,共6页
Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(P.falcipa... Objective:To analyse the rar gene repertoire and characterise the rhondroitin sulphate A (CSA)-binding activity of the Duffy-binding like(I)BI.) domains encoded by the var2csa gene of a Plasmodium falciparum(P.falciparum) isolate in Hainan Province,China.Methods:The sequences of var DBL1 regions were PCR-amplified,sequenced and the sequence characteristics was bioinformalically analysed.Recombinant proteins encoded by the var2csa genes were expressed and purified.The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays.Results:Fifty six unique DBI.a sequences were obtained,and the sequences represented similar diversity to the var genes of the genome parasite 3D7.There are two var2csa genes in the P.falciparum isolated from Hainan Province.Unlike in other falciparum parasites such as HB3,the two var2csa genes are more diverged.The receptor-binding capacity of DBL-5εand DBI.-6 e domains of HN var2CSA was studied.Conclusions:This work represented the diversity of rar genes of a P.falciparum isolate in China. 展开更多
关键词 MALARIA plasmodium falciparum antigenic variation VAR gene
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Effects of the configuration of a multi epitope chimeric malaria DNA vaccine on its antigenicity to mice 被引量:1
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作者 姜艳芳 林澄涛 +7 位作者 阴彬 何湘芸 毛映红 董敏 徐蓓 张连惠 刘宝丰 王恒 《Chinese Medical Journal》 SCIE CAS CSCD 1999年第8期14-18,共5页
Objective Four B and Th cell epitopes were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigeni... Objective Four B and Th cell epitopes were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigenicities were studied Methods The partially synthesized oligonucleotide was annealed, PCR amplified and cloned into a mammalian cell expression vector By using a pair of isocaudamers on the vector, different single copies of B epitopes were multiplied and were tenderly stringed into two groups of chimeric DNA vaccine with different configurations BALB/c mice were immunized with these DNA plasmids by either intramuscular or intradermal injections Results The antisera from the immunized mice tested by ELISA showed that only the configuration which had a single copy of universal T helper cell epitope, CS T3, located at the C terminal of the multi copy B cell epitopes induced a high antibody response The T helper cell epitope at any other position of the peptide, or the double T helper cell epitopes configured with the B cell epitopes did not enhance antibody response, and some configurations even decreased the humoral response to a B cell epitope Conclusion This study demonstrated that both combination and configuration of the epitope may affect the antigenicity of a chimeric multiple antigen 展开更多
关键词 malaria · plasmodium falciparum · epitopes · dna vaccine · isocaudamers
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恶性疟原虫重组质粒DNA接种诱导BALB/c小鼠的免疫应答 被引量:5
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作者 李学荣 余新炳 +2 位作者 罗树红 徐劲 陈观今 《中国寄生虫学与寄生虫病杂志》 CAS CSCD 北大核心 1998年第4期241-245,共5页
目的:观察重组质粒DNA直接免疫接种诱导BALB/c小鼠的免疫应答水平,为恶性疟原虫DNA疫苗在动物和人体的应用提供依据。方法:构建编码多价保护性抗原的重组质粒pcDNA3-Pf8,PCR法检测免疫鼠的肌肉、肝脏、肾... 目的:观察重组质粒DNA直接免疫接种诱导BALB/c小鼠的免疫应答水平,为恶性疟原虫DNA疫苗在动物和人体的应用提供依据。方法:构建编码多价保护性抗原的重组质粒pcDNA3-Pf8,PCR法检测免疫鼠的肌肉、肝脏、肾脏、心脏、脾脏和肺组织中pcDNA3-Pf8,ELISA、T淋巴细胞转化试验、体外抑制试验观察其诱导的体液免疫及细胞免疫水平。结果:用PCR法从上述组织中均检测到pcDNA3-Pf8,ELISA法测得免疫鼠血清的特异性抗体滴度达12560,淋巴细胞转化试验显示恶性疟原虫可溶性抗原能特异性地剌激免疫鼠脾细胞增殖,免疫血清在体外还能抑制恶性疟红内期疟原虫的生长、发育。结论:编码恶性疟原虫多价保护性抗原的重组质粒pcDNA3-Pf8直接免疫接种,能特异性地剌激BALB/c小鼠产生体液免疫和细胞免疫应答,其免疫血清在体外对疟原虫生长发育具有明显抑制作用。 展开更多
关键词 疟原虫 免疫应答 dna疫苗 重组质粒 dna
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细胞因子表达质粒对恶性疟原虫顶端膜抗原1DNA免疫的调节作用 被引量:3
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作者 李珣 缪军 +4 位作者 雷俊川 薛采芳 王宪锋 刘忠湘 李淑梅 《中国寄生虫学与寄生虫病杂志》 CAS CSCD 北大核心 2004年第3期136-138,共3页
目的 探讨细胞因子表达质粒对小鼠DNA免疫的促进和调节作用。 方法 构建编码恶性疟原虫顶端膜抗原 1(AMA1)完整胞外域的DNA免疫质粒VR10 2 0 /E ,构建编码小鼠细胞因子 (GM CSF)、白细胞介素 (IL)如IL 4和IL 12的真核表达质粒pcDNA3 ... 目的 探讨细胞因子表达质粒对小鼠DNA免疫的促进和调节作用。 方法 构建编码恶性疟原虫顶端膜抗原 1(AMA1)完整胞外域的DNA免疫质粒VR10 2 0 /E ,构建编码小鼠细胞因子 (GM CSF)、白细胞介素 (IL)如IL 4和IL 12的真核表达质粒pcDNA3 /GM CSF、pcDNA3 .1( ) /IL 4和pIL 12以及双顺反子质粒pGM CSF/pTPA E ,分组免疫小鼠 ,ELISA检测血清中特异性IgG及其亚类的水平 ,取小鼠脾细胞进行体外增殖。 结果  3种细胞因子质粒均有效增强了小鼠针对VR10 2 0 /E的免疫应答 ,抗体水平增加 7至 10倍 ,其中pcDNA3 /GM CSF质粒和pIL 12质粒分别显著促进了小鼠的IgG1和IgG2a应答 ,小鼠脾细胞的体外增殖水平亦有明显提高。 结论 利用编码GM CSF、IL 4和IL 12的表达质粒作为佐剂可有效增强小鼠针对AMA1DNA的免疫应答 ,并对免疫应答的类型产生调节作用。 展开更多
关键词 细胞因子表达 质粒 恶性疟原虫 顶端膜抗原1 dna免疫 调节作用 佐剂
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恶性疟原虫裂殖子表面蛋白1 DNA与改良痘苗病毒组合疫苗诱导小鼠抗体应答 被引量:3
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作者 李淑梅 李珣 +4 位作者 薛采芳 缪军 雷俊川 刘忠湘 王宪锋 《中国寄生虫学与寄生虫病杂志》 CAS CSCD 北大核心 2005年第2期93-96,共4页
目的探索DNA与改良痘苗病毒(MVA)组合免疫对增强恶性疟原虫裂殖子表面蛋白1(MSP1)抗体应答的作用。方法以人工合成MSP1全基因为基础分别构建DNA免疫质粒VR1020/190和重组MVA,单用VR1020/190或与表达质粒GM-CSF共同对小鼠进行初始免疫后... 目的探索DNA与改良痘苗病毒(MVA)组合免疫对增强恶性疟原虫裂殖子表面蛋白1(MSP1)抗体应答的作用。方法以人工合成MSP1全基因为基础分别构建DNA免疫质粒VR1020/190和重组MVA,单用VR1020/190或与表达质粒GM-CSF共同对小鼠进行初始免疫后,用重组病毒追加强化,采用DNA/MVA组合方案免疫BALB/c小鼠,ELISA测定血清IgG及其亚类水平,经腹腔接种转基因伯氏疟原虫Pb-PfM19进行攻击。结果DNA免疫能有效诱导小鼠产生抗MSP1-190抗体,其终点稀释度为1∶2500,GM-CSF质粒共免疫组抗体的终点稀释度为1∶11150,抗体亚类的测定表明GM-CSF质粒显著促进了IgG1类抗体应答,MVA追加可使单独免疫组和共免疫组抗体分别增加53和10倍;两实验组产生了水平相近的抗19000抗体(1∶32000),其含量占血清中MSP1总IgG的1/4-1/3。经转基因伯氏疟原虫Pb-PfM19攻击后小鼠的存活时间并没有明显延长(P>0.05)。结论采用合成MSP1全基因进行DNA/MVA组合免疫可诱导小鼠产生显著的抗体应答,抗体的详细特性和保护作用正在进一步研究中。 展开更多
关键词 恶性疟原虫裂殖子表面蛋白 抗体应答 痘苗病毒 疫苗诱导 组合 改良 BALB/c小鼠 ELISA测定 dna免疫 伯氏疟原虫 MSP1 血清IgG MVA CSF 免疫组 人工合成 表达质粒 重组病毒 IgG1 抗体亚类 总IgG 存活时间 MSPI 保护作用
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重组恶性疟原虫DNA质粒免疫小鼠及抗原表达的调控 被引量:6
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作者 谢文凯 陈启宇 +3 位作者 汪群斌 潘卫庆 颜日辉 陆德如 《生物工程学报》 CAS CSCD 北大核心 2000年第1期13-16,共4页
将恶性疟原虫MSP131基因序列,引入四环素(Tc)控制的真核表达载体pTRE,获得重组质粒pTRE31。将MSP131的原核表达载体pDS56T1转化大肠杆菌表达MSP131,亲和纯化后作检测用抗原。pTRE31与辅助质粒pTetoff(tTA)肌肉注射4周龄BALB/c小鼠,观察... 将恶性疟原虫MSP131基因序列,引入四环素(Tc)控制的真核表达载体pTRE,获得重组质粒pTRE31。将MSP131的原核表达载体pDS56T1转化大肠杆菌表达MSP131,亲和纯化后作检测用抗原。pTRE31与辅助质粒pTetoff(tTA)肌肉注射4周龄BALB/c小鼠,观察DNA介导免疫情况。结果显示,四环素饲喂的小鼠4周时血清抗体阳转率为71%(1/14),而不饲喂四环素组可达100%(14/14),表明用pTRE31/pTetoff重组质粒组合直接注射小鼠有效地引发了针对疟原虫MSP131抗原的体液免疫反应,且可受控于四环素。不饲喂四环素组小鼠在12周后仍能维持抗体阳性,倍比稀释ELISA显示血清抗体滴度在4周、8周和12周内持续上升。饲喂四环素组小鼠4周后停止饲喂Tc,第8周和第12周检测仍有部分(60%)小鼠血清抗体阳转,而继续饲喂Tc的小鼠未有抗体阳转,暗示重组质粒DNA在小鼠体内可持续存在至少4周并仍具备表达功能。 展开更多
关键词 恶性疟原虫 dna疫苗 四环素 Tet-off调控系统
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荧光蛋白报告载体观察刚地弓形虫SAGl、MIC3、ROP2鸡尾酒DNA疫苗的表达及其免疫原性的研究 被引量:6
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作者 王燕娟 曹建平 +2 位作者 孙雅雯 徐馀信 沈玉娟 《中国寄生虫学与寄生虫病杂志》 CAS CSCD 北大核心 2015年第5期368-371,376,共5页
目的构建能同时表达多个基因的刚地弓形虫DNA鸡尾酒疫苗并初步观察其免疫原性。方法从基因组DNA扩增刚地弓形虫表面抗原(surface antigen,SAG)、微线体蛋白(microneme,MIC)和棒状体蛋白(rhoptry protein,ROP)基因片段,克隆到真核荧光表... 目的构建能同时表达多个基因的刚地弓形虫DNA鸡尾酒疫苗并初步观察其免疫原性。方法从基因组DNA扩增刚地弓形虫表面抗原(surface antigen,SAG)、微线体蛋白(microneme,MIC)和棒状体蛋白(rhoptry protein,ROP)基因片段,克隆到真核荧光表达载体pShuttle-CMV-MCS-EF1α-Am Cyan,pLVX-IRES-Zsgreen及pLVX-IRES-rfp中,构建pShuttle-SAG1,pLVX-Zsgreen-MIC3和pLVX-rfp-ROP2表达质粒。通过聚乙烯亚胺法用混合质粒转染293F细胞48 h,荧光显微镜下观察3个基因的表达情况。将30只C57BL/6雄性小鼠随机分为A、B、C组,分别肌内注射生理盐水(50μl)、pShuttle+pLVX-Zsgreen+pLVX-rfp混合空质粒(2μg/μl,各17μl)、pShuttle-SAG1+pLVX-Zsgreen-MIC3+pLVX-rfp-ROP2混合重组质粒(2μg/μl,各17μl)。免疫28 d后,ELISA检测血清抗刚地弓形虫IgG抗体水平,初步评价该疫苗的免疫原性。结果从刚地弓形虫基因组成功扩增出1、1.1及1.7 kb的SAG1、MIC3和ROP2序列。成功构建了pShuttle-SAG1、pLVX-Zsgreen-MIC3和pLVX-rfp-ROP2真核荧光表达质粒,转染293F细胞后观察到相应的蓝、绿、红报告荧光。免疫小鼠后28 d,ELISA测得A、B、C组IgG抗体的吸光度(A450值)分别为(0.620±0.029)、(0.741±0.040)、(1.561±0.131),C组显著高于A、B组(P<0.01)。结论刚地弓形虫SAG1、MIC3和ROP2基因混合重组质粒组成的DNA鸡尾酒疫苗能诱导小鼠产生良好的免疫原性,且多个荧光蛋白真核表达载体能够较好地指示目的基因的表达。 展开更多
关键词 刚地弓形虫 基因重组 表面抗原 微线体蛋白 棒状体蛋白 鸡尾酒疫苗 免疫原性
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结核分枝杆菌四价DNA疫苗免疫原性和保护效率研究 被引量:6
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作者 顾田园 蔡宏 +2 位作者 田霞 余大海 朱玉贤 《生物化学与生物物理进展》 SCIE CAS CSCD 北大核心 2005年第4期347-352,共6页
对结核杆菌四价DNA疫苗的免疫应答和保护效果进行了评价.用编码结核分枝杆菌Ag85B、MPT64、MPT70和PstS-3等4种抗原蛋白的基因分别构建的单价DNA疫苗混合成四价苗免疫小鼠.3次免疫后21天,四种抗原特异性抗体滴度分别达到1:6 400、1:51 ... 对结核杆菌四价DNA疫苗的免疫应答和保护效果进行了评价.用编码结核分枝杆菌Ag85B、MPT64、MPT70和PstS-3等4种抗原蛋白的基因分别构建的单价DNA疫苗混合成四价苗免疫小鼠.3次免疫后21天,四种抗原特异性抗体滴度分别达到1:6 400、1:51 200、1:6 400、1:6 400.四种蛋白质均能诱导脾脏细胞产生较高水平的抗原特异性IFN-γ,浓度分别为10 582.14 ng/L、13 635.97 ng/L、14 213.15 ng/L和9 657.35 ng/L.三次免疫后经静脉强毒攻击,四价苗组小鼠肺脏和脾脏的载菌数分别减少到阴性组的1/650和1/130.对肺组织的病理形态特征观察表明,空载体免疫的小鼠肺部严重损伤,肺实质干酪样坏死,坏死结节占肺实质的70%~80%,而四价苗免疫的小鼠,肺组织结构正常,肺泡轮廓清晰.研究首次证实,Ag85B、MPT64、MPT70和PstS-3 4种结核杆菌抗原蛋白编码基因组成的四价DNA疫苗,具有很高的免疫应答水平和保护效率. 展开更多
关键词 结核分枝杆菌 抗原蛋白 四价dna疫苗 免疫原性 保护效率
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弓形虫多表位抗原基因DNA疫苗对BALB/c小鼠的免疫保护性研究 被引量:3
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作者 杨培梁 陈晓光 +3 位作者 李华 周晓红 彭鸿娟 吴焜 《中国病原生物学杂志》 CSCD 2007年第5期340-343,共4页
目的以弓形虫多表位抗原基因DNA疫苗免疫小鼠,评价该疫苗对弓形虫感染产生的免疫保护作用。方法利用PCR技术和亚克隆技术,构建弓形虫多表位抗原基因真核表达重组质粒pcDNA3-MAG,以质粒纯化试剂盒大量制备质粒,同时分别以载体质粒pcDNA3... 目的以弓形虫多表位抗原基因DNA疫苗免疫小鼠,评价该疫苗对弓形虫感染产生的免疫保护作用。方法利用PCR技术和亚克隆技术,构建弓形虫多表位抗原基因真核表达重组质粒pcDNA3-MAG,以质粒纯化试剂盒大量制备质粒,同时分别以载体质粒pcDNA3和PBS液为空质粒对照和空白对照,与lipofectin按5∶2的比例混合后,经股四头肌注射免疫小鼠,间隔两周,连续免疫3次,通过检测小鼠血清中特异的IgG抗体、IFN-γ和IL-4含量,评价疫苗产生的体液免疫和细胞免疫水平。以强毒型RH株弓形虫感染免疫小鼠,统计小鼠的存活时间,评价疫苗产生的免疫保护性。结果经双酶切及DNA测序鉴定,所构建的重组质粒pcDNA3-MAG读码框架正确。与免疫前及载体质粒和空白对照组相比,小鼠免疫后产生特异性IgG抗体,并引发高水平IFN-γ。攻虫后,实验组较对照组小鼠存活时间明显延长。结论弓形虫多表位抗原基因DNA能诱导BALB/c系小鼠产生特异的细胞免疫和体液免疫,对弓形虫感染可产生一定的免疫保护性。 展开更多
关键词 弓形虫 多表位抗原 dna疫苗 免疫
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基因疫苗pcDNA3.0-PSMA肿瘤细胞模型的建立 被引量:2
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作者 汪波 曹开源 +4 位作者 徐向东 徐霖 袁广卿 张甜 丘少鹏 《热带医学杂志》 CAS 2008年第7期639-641,656,F0003,共5页
目的构建表达前列腺特异性膜抗原(PSMA)的肿瘤细胞模型,为基因疫苗的抑瘤效应研究及免疫机制的探讨提供实验材料。方法脂质体转染法分别将PSMA-pcDNA3.0质粒和pcDNA3.0质粒转染至SP2/0细胞,G418筛选后获得了稳定生长的阳性克隆细胞株,RT... 目的构建表达前列腺特异性膜抗原(PSMA)的肿瘤细胞模型,为基因疫苗的抑瘤效应研究及免疫机制的探讨提供实验材料。方法脂质体转染法分别将PSMA-pcDNA3.0质粒和pcDNA3.0质粒转染至SP2/0细胞,G418筛选后获得了稳定生长的阳性克隆细胞株,RT-PCR、间接免疫荧光法、Westernblot检测PSMA蛋白的表达。结果RT-PCR、间接免疫荧光法和Western blot均证明转染了PSMA-pcDNA3.0质粒的SP2/0细胞表达PSMA。结论成功建立了稳定表达PSMA的小鼠肿瘤细胞模型,最终为前列腺癌的预防和免疫治疗研究打下了坚实的基础。 展开更多
关键词 前列腺特异性膜抗原 基因疫苗 基因表达
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恶性疟原虫DNA疫苗在小鼠体内组织分布和安全性的初步研究 被引量:1
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作者 郑春福 吴少庭 +2 位作者 陈雅棠 高世同 林敏 《中国人兽共患病杂志》 CSCD 北大核心 2002年第6期29-31,共3页
目的 探讨疟疾DNA疫苗在小鼠体内的组织分布 ,并对其安全性进行观察。方法 将重组质粒pBK -CSP经肌肉途径免疫BALB/c小鼠 ,分别在 4周和 8周后剖杀动物并摘取各种组织 ,抽提全组织DNA进行PCR扩增后经琼脂糖凝胶电泳分析 ,并对DNA疫苗... 目的 探讨疟疾DNA疫苗在小鼠体内的组织分布 ,并对其安全性进行观察。方法 将重组质粒pBK -CSP经肌肉途径免疫BALB/c小鼠 ,分别在 4周和 8周后剖杀动物并摘取各种组织 ,抽提全组织DNA进行PCR扩增后经琼脂糖凝胶电泳分析 ,并对DNA疫苗的安全性进行观察。结果 免疫 4周和 8周后 ,仅有DNA疫苗接种位点的肌肉组织检测到CSP基因 ,而 10 0 μg的质粒DNA并未产生明显的毒副作用。 结论 疟疾DNA疫苗接种 4周后 ,质粒DNA仅分布于接种部位 ,并可持续至 8周以上 ,而未发现毒副作用。 展开更多
关键词 恶性疟原虫 dna疫苗 组织分布 安全性 疟疾
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重组恶性疟原虫DNA质粒免疫小鼠制备单克隆抗体 被引量:1
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作者 谢文凯 钟平 +3 位作者 潘卫庆 陈启宇 汪群斌 陆德如 《微生物学报》 CAS CSCD 北大核心 2000年第4期389-393,共5页
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PEG1 450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... 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PEG1 450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∶1 0 0 0 0。经免疫球蛋白类型和亚类鉴定 ,2株杂交瘤细胞株均为IgG1 。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。 展开更多
关键词 严重疟原虫 dna疫苗 单克隆抗体 制备 载体dna
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恶性疟原虫复合抗原DNA疫苗诱导小鼠的细胞免疫及体液免疫应答 被引量:2
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作者 姚朗 李英杰 李明 《中国寄生虫病防治杂志》 CSCD 2002年第6期342-345,共4页
目的 观察用恶性疟原虫复合抗原基因 HGFSP构建的 DNA疫苗 pc- HGFSP免疫小鼠诱导的细胞及体液免疫应答。 方法 用 pc- HGFSP肌注免疫 C5 7BL/6小鼠并加强免疫 2次 ,取小鼠脾淋巴细胞及血清测定 :脾淋巴细胞增殖活性 (MTT法 ) ;NK细... 目的 观察用恶性疟原虫复合抗原基因 HGFSP构建的 DNA疫苗 pc- HGFSP免疫小鼠诱导的细胞及体液免疫应答。 方法 用 pc- HGFSP肌注免疫 C5 7BL/6小鼠并加强免疫 2次 ,取小鼠脾淋巴细胞及血清测定 :脾淋巴细胞增殖活性 (MTT法 ) ;NK细胞活性和 CTL活性 (L DH法 ) ;脾脏 CD4 +及 CD8+ T细胞亚群 (免疫荧光法 ) ;血清 pc- HGFSP抗原特异性抗体 (EL ISA法 )及一氧化氮 (NO)含量。  结果 与 pc DNA3对照比较 ,pc- HGFSP免疫小鼠脾淋巴细胞增殖活性增高 2 4 %~ 37% ;NK细胞活性增高 38%~ 90 % ;CTL 活性增高 6 5 %~ 15 3% ;CD8+ T细胞亚群增加。免疫血清产生HGFSP抗原特异性 Ig G抗体 ;NO含量也有所增高。 结论 恶性疟 DNA疫苗 pc- HGFSP有一定的诱导小鼠细胞免疫和体液免疫应答的作用。 展开更多
关键词 疟原虫 恶性 dna疫苗 抗原 免疫
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恶性疟原虫FCC-1/HN株DNA疫苗在小鼠体内的免疫反应 被引量:1
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作者 吕芳丽 余新炳 +1 位作者 郭虹 陈观今 《中山大学学报(医学科学版)》 CAS CSCD 2000年第S1期1-5,共5页
:【目的】探讨恶性疟原虫DNA疫苗在小鼠体内的免疫反应。【方法】将恶性疟原虫FCC 1/HN株重组质粒pcDNA3 Pfs2 5及 pcDNA3 EBA175 /HRPⅡ经骨骼肌途径分别单独注射或两者混合注射免疫BALB/c小鼠 ,观察免疫后不同时间点血清中IgG抗体滴... :【目的】探讨恶性疟原虫DNA疫苗在小鼠体内的免疫反应。【方法】将恶性疟原虫FCC 1/HN株重组质粒pcDNA3 Pfs2 5及 pcDNA3 EBA175 /HRPⅡ经骨骼肌途径分别单独注射或两者混合注射免疫BALB/c小鼠 ,观察免疫后不同时间点血清中IgG抗体滴度、脾淋巴细胞增殖反应、CD4+/CD8+T细胞亚群比值和NK细胞杀伤活性的变化。【结果】pcD NA3 EBA175 /HRPⅡ与 pcDNA3 Pfs2 5分别单独肌肉注射或两者混合肌肉注射免疫小鼠后 ,均可见血清IgG抗体滴度增高 ;针对恶性疟原虫抗原的特异性T淋巴细胞增殖反应增强 ;CD4+/CD8+T细胞比率下降以及NK细胞杀伤活性增强。【结论】提示肌肉注射为一有效的DNA疫苗免疫途径 ,采用编码恶性疟原虫有性期阶段或无性红细胞阶段的重组质粒单独或两者混合免疫小鼠 ,均能诱导明显的体液免疫反应、细胞免疫反应和NK细胞杀伤活性。 展开更多
关键词 疟原虫 恶性 疫苗 dna 接种 杀伤细胞 天然/免疫学
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