恶性疟原虫丝氨酸重复抗原部分基因的克隆及其表达

采用PCR方法特异性扩增恶性疟原虫云南株(PFD-3/YN)丝氨酸重复抗原基因片段,经基因序列测定后克隆于pWR450-1融合蛋白表达载体,并转化大肠杆菌TG1。在不同菌体浓度及不同剂量IPTG诱导下检测SERA融合蛋白的表达。采用dot-ELISA及Western-blot分析对表达产物进行鉴定,结果显示SERA基因与pWR450-1重组后在大肠杆菌TG1中表达一72kDa的融合蛋白,当工程菌OD590值为0.8～1.2时,加入终浓度1mmol/LIPTG进行诱导,表达量较高。dot-ELISA和Westernblot分析表明SERA基因表达产物能被抗SERA单克隆抗体所识别。     

恶性疟原虫复制蛋白PfRPA2的原核表达及多抗制备

目的克隆表达恶性疟原虫复制蛋白PfRPA2亚基,制备多抗,为该蛋白的功能研究奠定基础。方法PCR扩增目的基因片段,克隆到表达载体pGEX．KG中,构建PjRPA2／pGEX-KG原核表达载体,IPTG诱导表达,SDS—PAGE电泳分析表达产物．谷胱甘肽柱纯化蛋白,Western blot检测其抗原性,以纯化的GST-PfRPA2免疫小鼠,制备抗tyRPA2的多抗．间接ELISA法检测鼠血清效价,Western blot鉴定多抗特异性。结果成功构建了重组踯PA2／pGEX．KG原核表达质粒,并在大肠杆菌中以可济眭形式高效表达,纯化表达产物,制备抗邸PA2的鼠多抗,效价为10-7,Western blot证实此抗体可识别恶性疟原虫内与PJRPA2蛋白位置对应的特异性条带。结论恶性疟原虫复制蛋白PfRPA2亚基在大肠杆菌中获得可溶性高效表达,纯化表达产物能诱导小鼠生产能识别天然蛋白的特异性抗体。     

大鼠透明质酸合成酶-2抗血清的制备及鉴定

目的: 制备大鼠透明质酸合成酶-2(HAS-2)抗血清并鉴定抗血清灵敏度及其特异性.方法: 通过生物信息学分析选取HAS-2非保守的亲水区域作为抗原, 通过原核表达纯化后免疫家兔, 制备出抗血清.Western blot检测所制备抗血清的灵敏度.真核瞬时表达大鼠HAS-2和HAS-3, 分别用抗His抗体和抗血清检测, 鉴定制备抗血清的特异性.结果: 通过原核表达并纯化得到抗原多肽, 免疫家兔后获得ELISA效价大于1∶ 10 000的抗血清.所制备的抗血清至少可以检测出10 ng以上的抗原多肽, 并且能够特异地检测HAS-2而与HAS-3无反应.结论: 成功通过大鼠HAS-2非保守区域多肽的原核表达产物制备出高灵敏度和特异性的大鼠HAS-2抗血清, 为研究透明质酸(HA)的病理生理意义提供了有利支持.     

单纯疱疹病毒Ⅰ形胸苷激酶基因在大肠杆菌中的融合表达

用EcoRⅠ和HindⅢ双酶切已构建的含单纯疱疹病毒I型胸苷激酶(HSV-1TK)基因的pUC18/TK质粒,将切出的TK基因片段克隆入原核表达载体pWR450-1中,构建成HSV-1TK基因重组表达质粒pWR450-1/TK。以HSV-1TK特异性引物TK1For和TK2Rev进行PCR鉴定可扩增出预期的503bp的TK基因编码区部分序列;EcoRⅠ和HindⅢ双酶切质粒pWR450-1/TK,可切出约1150bp的DNA片段,表明重组质粒中已插入HSV-1TK基因片段。用IPTG诱导pWR450-1/TK/JM109,表达出相对分子质量（Mr）约为97000的TK和β-半乳糖苷酶(Mr55000)融合蛋白。薄层扫描显示,该融合蛋白含量占菌体蛋白总量的27.47％。     

福氏志贺菌毒力蛋白IpaC的表达、纯化及免疫活性鉴定

目的表达、纯化福氏志贺菌毒力蛋白IpaC并对其免疫活性进行鉴定。方法将含有ipaC基因的pET32a．ipaC表达质粒载体转入大肠杆菌BL21（kDE3）中表达，表达产物进行SDS—PAGE鉴定，并采用QIA expressionist^TM蛋白纯化系统纯化IpaC蛋白；用纯化的蛋白免疫动物获得抗血清，Western-blot检测抗原的免疫活性。结果诱导后的表达产物经SDS—PAGE发现有一相对分子量约为63000的条带，其含量约占总蛋白量的11％，对融合蛋白进行纯化纯度可达90％以上，抗His的抗体Western-blot分析，发现特异性区带出现在63000u处，证明该蛋白是所要表达的融合蛋白，制备的免疫血清可与Ipac发生特异免疫反应。结论pET32a-ipaC重组表达质粒转入大肠杆菌后，可稳定、高效地表达毒力蛋白IpaC，且纯化后的蛋白具有免疫活性。     

恶性疟原虫抗原基因在鼠伤寒沙门氏菌中的表达及免疫原性的初步鉴定

将人工合成的恶性疟原虫杂合７４肽抗原基因克隆到表达载体ｐＷＲ４５０－１中，获得了重组质粒ｐＷＲＣ，将其先转化到减毒伤寒沙门氏菌ＬＢ５０００修饰后，再转化到ＳＬ３２６１，得到重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲＣ）。ｐＷＲＣ在ＳＬ３２６１中以β－半乳糖苷酶－杂合多肽抗原Ｃ融合蛋白（ＧＺ－Ｃ）形式表达，表达量约占菌体蛋白总量的１１．３％．Ｗｅｓｔｅｒｎｂｌｏｔ及免疫双扩散显示ＧＺ－Ｃ可与兔抗ＧＺ－Ｃ抗原的免疫血清发生特异反应。ＧＺ－Ｃ识别兔抗ＧＺ－Ｃ及鼠抗恶性疟原虫抗血清的ＥＬＩＳＡ滴度分别为１：３２００及１：５１２０。初步结果表明ＳＬ３２６１（ｐＷＲＣ）表达的ＧＺ－Ｃ具有抗原性。连续传代ＳＬ３２６１（ｐＷＲＣ）未见质粒ｐＷＲＣ丢失及对宿主有明显的毒性。     

丙型肝炎病毒及恶性疟原虫复合抗原基因表达及诱导小鼠免疫应 …

将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ及恶性疟原虫（Ｐｆ）多价抗原基因ＡＢ（ＳＰｆ６６＋ＳＰｆ１０５）克隆到表达载体ｐ＾ＷＲ４５０－１中,以β－半乳糖甘酶（ＧＺ）－ＨＣＶ－Ｐｆ复合多肽抗原的形式在大肠杆菌中高效融合表达了目的蛋白ＧＺ－ＣＡＢ,表达量约３０％。所表达的重组抗原可被ＨＣＶ患者血清及鼠抗Ｐｆ抗体特异识别,显示了该融合蛋白质具有ＨＣＶ和Ｐｆ的免疫原性。该蛋白免疫小鼠后,诱发     

恶性疟原虫红细胞表面膜蛋白1DBLα区不同区段与血型抗原的结合特性分析

根据大肠杆菌密码子组成特点,重新优化并合成FCR3S1.2株恶性疟原虫红细胞表膜蛋白1(PfEMP1)DBLα区基因序列,利用PCR的方法,将优化后的基因序列分为3段.分别将全长基因和3个基因片段在大肠杆菌中表达、纯化.通过重组蛋白与血型抗原的亲和试验,分析功能区与血型抗原是否具有亲和力.结果表明,重组的PfEMP1-...     

恶性疟原虫FCC—1／HN株环子孢子蛋白基因分枝杆菌——大肠杆穿梭表达重组质粒的构建及序列测定

本文对恶性疟原虫环子孢子蛋白(circumsporozoiteprotein,CSP)基因片段进行克隆和序列测定。根据恶性疟原虫837株基因编码序列设计合成一对引物,采用PCR技术从恶性疟原虫FCC-1/HN株基因组DNA中特异扩增CSP基因片段的Ⅰ区、中央重复区、重复区后可变区和Ⅱ区;经纯化的扩增产物用BamHⅠ和KpnⅠ双酶切后,定向克隆入大肠杆菌——分枝杆菌穿梭表达质粒,转化感受态大肠杆菌DH5α,重组克隆经抗性筛选和快速凝胶电泳鉴定,再经PCR和酶切鉴定,并对重组子进行序列测定。结果表明从恶性疟原虫FCC-1／HN株基因组DNA中可特异扩增出约1171bp的基因片段,阳性重组质粒经双酶切和PCR鉴定与预期的结果一致,序列测定表明所克隆的基因和编码环子孢子抗原的基因片段相符。     

重组霍乱毒素B亚基与恶性疟原虫多表位融合蛋白的免疫原性研究

目的　评价霍乱毒素B亚基与恶性疟原虫多价抗原的融合蛋白的免疫原性。方法　在大肠杆菌中表达重组霍乱毒素与恶性疟原虫多价抗原的融合蛋白 ,通过免疫小鼠评价融合蛋白的免疫原性。结果　通过亲和层析纯化霍乱毒素B亚基与恶性疟原虫多价抗原的融合蛋白 ,不加任何佐剂 ,以 5 0 μg/只的剂量免疫C5 7BL/ 6J小鼠 ,3次免疫后 ,CTB抗体滴度达 1∶6 40 0 ,抗疟原虫抗体滴度 1∶16 0 0 ,其CTL活性为 2 0 7%。结论　霍乱毒素具有较好的佐剂作用 ,经融合蛋白免疫的小鼠能产生良好的体液和细胞免疫 ,为评价该抗原的免疫保护作用打下了基础     

恶性疟原虫环子孢子蛋白单链抗体与按蚊cecropin A融合基因的构建、原核表达及生物学活性的初步分析

根据按蚊偏嗜性密码子对鼠源性恶性疟原虫环子孢子蛋白单链抗体2a10基因进行改造,并融合按蚊抗菌肽cecropin A编码基因。将获得的cecrop-m2a10融合基因克隆入原核表达载体pET32a（＋）中,对表达产物进行SDS-PAGE、Western-blot分析并利用琼脂糖扩散法检测其抗菌活性。结果对鼠源性环子孢子蛋白单链抗体2a10中6种氨基酸的170个核苷酸进行了改造,融合cecropinA编码基因,成功构建了cecrop-m2a10融合基因。靶基因在大肠杆菌中以融合蛋白和包涵体的形式高效表达,包涵体经尿素溶解变性及透析复性后表达蛋白的纯度达75%并具备抗大肠杆菌DH5α的活性。本实验为进一步研究cecrop-m2a10在转基因蚊中的多重抗病原效应提供了基础。     

人La多肽融合蛋白在E.coli中的表达

目的　构建在E .coli中表达人La多肽的质粒。方法以人脾cDNA为模板 ,通过PCR获得La多肽 (全长 )的DNA片段。将此片段利用双酶切定向克隆到MBP(麦芽糖结合蛋白 )融合系统的载体pMALTM c中 ,表达可溶性融合蛋白 ,并通过亲和层析予以纯化。结果免疫印迹实验 (IBT)证明 ,表达的融合蛋白具有La抗原多肽的特异性 ,而敏感性超过生化制备的ENA制品。结论重组融合蛋白与天然ENA制品存在免疫学同一性     

Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein.

From a Plasmodium falciparum cDNA expression library derived from mRNA of the asexual blood stages, we isolated and sequenced five different cDNA clones whose predicted protein products were unusually rich in asparagine (Asn). Two of the clones, R5 and G5, contain tandem imperfectly repeated sequences based on Asn-Asn-Thr (NNT) and Asn-Asn-Met (NNM) respectively. The other three, E4, C5 and R13, as well as G5, contain stretches of polyasparagine varying in length from 2 to 26 residues. Results of DNA blotting experiments with the individual cDNA sequences as probes suggest that each of the five clones corresponds to a different P. falciparum gene. The fragments of P. falciparum proteins expressed by the cDNA clones shared cross-reactive antigenic determinants which were present on multiple P. falciparum proteins. In immunoblotting experiments, owl monkey antibodies selected for binding to the polypeptide expressed by clone E4, C5 or G5 reacted with the expressed proteins from all 5 clones, and with at least 10 proteins from schizont infected erythrocytes. The cross-reactive epitopes could be modeled by two Asn-rich peptide structures: (1) (NNT)8, whose sequence was based on the R5 repeat; and (2) (NPNA)6, whose sequence was based on the Asn-rich repeat of the P. falciparum circumsporozoite protein (CSP). Antibodies that bound to each peptide were selected from sera of immune monkeys that had never been exposed to sporozoites. The selected antibodies bound all 5 expressed proteins in immunoblotting assays and also bound to several proteins from parasitized erythrocytes. Such cross reactivity between the CSP repeating unit and several blood-stage antigens has not been previously reported.     

A Plasmodium falciparum malaria vaccine candidate which contains epitopes from the circumsporozoite protein and a blood stage antigen, 5.1.

The previously described Plasmodium falciparum blood stage antigen, 5.1 (also referred to as exp-1) was expressed at a high level in Escherichia coli. Saimiri monkeys immunised with purified recombinant antigen 5.1 were partially protected from P. falciparum blood stage parasite challenge. The gene coding for 5.1 was combined with DNA coding for an (Asn-Ala-Asn-Pro)19 sequence (abbreviated (NANP)19 in the one-letter amino acid code). To facilitate purification of the recombinant protein, DNA coding for a hexahistidine (His6) sequence was introduced at the 5' end of the gene (proteins containing His6 have high affinity for Ni(2+)-chelate columns even in the presence of 6 M guanidine HCl). The recombinant protein, His6-5.1-(NANP)19 with an apparent molecular size of 40 kDa could be highly purified by a combination of 4 steps: (1) release and solubilization of the recombinant fusion protein from E. coli in the presence of 6 M guanidine-HCl; (2) precipitation of over 60% of the bacterial proteins by the addition of ammonium sulphate to 50% saturation; (3) affinity chromatography on a Ni(2+)-chelate column in the presence of 6 M guanidine-HCl; (4) adsorption onto a cation exchange resin in the presence of 6 M urea, and elution with an increasing NaCl gradient. Compared with the previously tested tetanus toxoid-(NANP)3 malaria vaccine, this protein elicits an anti-(NANP)n response which more closely resembles that evoked by native sporozoites. The recombinant vaccine also induces the production of antibodies against the blood stages of the malaria parasite.     

Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein.

This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactosidase fusion protein and used as antigen after purification and biotinylation. Specific IgM antibodies were found in 51% (39/77) of sera from adult Liberians immune to malaria. The binding of IgM antibodies was specific for the malaria portion of the fusion protein and no cross-reactivity was found in sera from patients with IgM antibodies due to other diseases. Inhibition studies with a fusion protein containing amino acid residues 816-1134 (GLURP816-1134) representing the carboxy-terminal repeat region suggested a different use of epitopes for IgM antibodies in different individuals.     

Characterization and vaccine potential of a novel recombinant coccidial antigen.

A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites. The cDNA fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gt11, transferred to a plasmid, and introduced into Escherichia coli for analysis of the gene products. The strain carrying the plasmid produced GX3262 as part of a fusion protein consisting of the first 1,006 amino acids of E. coli beta-galactosidase and 112 amino acids of the E. tenella protein of approximately 12 kilodaltons. Partially purified antigen, heat-killed recombinant bacterin, and live E. coli containing the recombinant coccidial antigen were used to immunize 1-week-old or newly hatched broiler chicks. Several immunization protocols were utilized, including boosts with partially purified beta-galactosidase-GX3262, bacterin, or small numbers of live E. tenella oocysts. After challenge with an experimental E. tenella infection, the birds were evaluated by scoring cecal lesions to determine the level of protection. The greatest degree of protection was seen after only a single immunization of 2-day-old birds with a live recombinant E. coli preparation. The results presented here identify GX3262 as a potential candidate coccidial vaccine antigen and provide evidence for the first time that newly hatched chickens can be successfully vaccinated with a recombinant antigen.     

丙型肝炎病毒及恶性疟原虫复合抗原基因表达及诱导小鼠免疫应答的研究

将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ及恶性疟原虫（Ｐｆ）多价抗原基因ＡＢ（ＳＰｆ６６＋ＳＰｆ１０５）克隆到表达载体ｐＷＲ４５０ ｌ中,以β 半乳糖甘酶（ＧＺ） ＨＣＶ Ｐｆ复合多肽抗原的形式在大肠杆菌中高效融合表达了目的蛋白ＧＺ ＣＡＢ,表达量约３０％。所表达的重组抗原可被ＨＣＶ患者血清及鼠抗Ｐｆ抗体特异识别,显示了该融合蛋白质具有ＨＣＶ和Ｐｆ的免疫原性。该蛋白免疫小鼠后,诱发针对ＧＺ ＣＡＢ、ＧＺ ＰＣＸ及ＰｆＡｇ的特异性体液免疫应答及针对ＧＺ ＣＡＢ、ＧＺ ＰＣＸ的迟发性超敏反应,ＣＤ８＋Ｔ细胞明显升高,提示该复合抗原作为ＨＣＶ Ｐｆ双价疫苗的可行性。     

抗人CD33单链抗体的基因构建、表达及其生物活性检测

目的：构建及表达抗人CD33单链抗体（抗CD33-scFv）基因，并检测其生物活性。方法：采用RT—PCR方法从分泌抗人类白细胞表面分化抗原CD33单克隆抗体（mAb）的杂交瘤细胞中克隆出VL和VH可变区基因，再通过重叠延伸拼接（splice—overlap extension）PCR方法在VH和VL可变区基因之间引入柔性连接肽（Gly4Ser）3，体外构建抗人CD33-scFv基因。将其克隆至原核表达载体PET-28a（＋）并在大肠杆菌Rosetta（DE3）中表达。结果：SDS-PAGE和Western blot分析结果表明，抗CD33-scFv在Rosetta（DE3）菌中获得高效表达，重组蛋白的相对分子质量（Mr）为30000，表达产物以不溶性包涵体形式存在，经过溶解包涵体，镍柱亲和层析纯化和体外复性过程，获得了高纯度的scFv片段。流式细胞术（FCM）分析结果证实抗CD33-scFv可与人类白细胞表面的分化抗原CD33结合，保留了鼠源性mAb的与CD33结合的活性。结论：重组抗人CD33-scFv基因构建与表达成功，并且通过复性得到有生物活性的scFv，为下一步针对髓系恶性肿瘤的靶向治疗奠定了基础。     

Antigenicity and immunogenicity of recombinant glutamate-rich protein of Plasmodium falciparum expressed in Escherichia coli.

A recombinant Plasmodium falciparum glutamate-rich protein (GLURP) was produced in Escherichia coli as a nearly full-length protein. In order to map immunodominant regions on GLURP, the nonrepetitive amino-terminal region (R0) as well as the central repeat region (R1) and the carboxy-terminal repeat region (R2) were also produced as separate products. All four purified gene products reacted specifically with serum samples from adults living in an area of Liberia where malaria is holoendemic. It appears that the human immune response against GLURP is primarily directed against the R2 region because 94% of the serum samples reacted with this region in an immunoassay. Antibody reactivity against the R0 region was also observed in 75% of the serum samples, while the R1 region showed only weak antibody-binding activity. When the nearly full-length GLURP molecule was adsorbed to Al(OH)3 it was found to be immunogenic in mice. In these experiments, the antibody response was almost exclusively directed against the R2 region. When anti-GLURP sera were obtained from rabbits immunized with the three regions, R0, R1, and R2, respectively, they recognized in immunoprecipitation experiments authentic GLURP from P. falciparum grown in vitro. These results demonstrate that GLURP produced in E. coli can induce a humoral immune response against GLURP derived from blood-stage parasites.     

Vaccines against enterotoxigenic bacterial pathogens based on hybrid Salmonella that express heterologous antigens

In this report, we examine two aspects in the development of a vaccine against enterotoxigenic bacterial pathogens based on hybrid Salmonella that express heterologous antigens. First, we describe the construction of a non-toxic fusion peptide for immunization against Escherichia coli that produce heat-labile (LT) and heat-stable (ST) enterotoxins. For that construction, the 5' terminus of the gene coding for ST was fused to the 3' terminus of the gene coding for the binding subunit of LT(LT-B). The ST gene was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion. Maximum expression of ST antigenicity was obtained when a seven-amino-acid proline-containing linker was included between the LT-B and ST moieties. The purified LT-B/ST fusion peptide consisted of a single polypeptide chain with an apparent molecular weight of 18,000. The LT-B/ST fusion peptide was non-toxic and immunologic determinants of both LT and ST were recognized by antibodies directed against the native toxins. Animals immunized with either crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST. The second aspect reported here examines a mechanism for stabilizing expression of heterologous antigens in attenuated Salmonella mutants by integration of the heterologous gene (LT-B) into the chromosome of the carrier. A comparative in vitro study of the levels of expression of LT-B between the cointegrate strain and an isogenic strain carrying the LT-B gene on a multicopy plasmid demonstrated that the initial levels of expression of both strains is similar, that the plasmid-carrying strain loses the ability to express the heterologous antigen very quickly and that the cointegrate continues to maintain and express the antigen without the requirement for a stabilizing antibiotic.