采用简并引物克隆葎草花粉过敏原全长同源cDNA

目的：建立一套稳定可靠的方法，对过敏原性物种中的过敏原同源基因进行快速克隆。方法：在分析生物信息数据库中积累的大量过敏原序列同源性的基础上，设计简并引物，基于高质量Lǘ草花粉RNA，逆转录合成cDNA。采用Touchdown方式在cDNA池中进行选择性PCR扩增。同时借助梯度PCR程序，对引物扩增的简并性作进一步强化，并结合RACE技术获取全长cDNA．进而对Lǘ草花粉中的过敏原同源基因进行克隆。结果：成功地获得3个全长cDNA克隆。序列分析显示．这些基因与已知过敏原的基因序列相似性高达79％-85％，初步认定其为泛过敏原肌球蛋白抑制蛋白(profilin)的同源基因。对比RACE技术获得的相应基因的全长序列发现，这些序列在引物结合处与简并引物序列之间存在4个碱基的差异，提示采用Touchdown方式的梯度PCR程序．可使引物的简并性得到进一步扩展。结论：简并引物与Touchdown梯度PCR相结合的方法．能够对以Lǘ草为代表的基因组未曾深入研究的物种中的过敏原同源基因进行有效克隆.     

嵌合基因在肿瘤发生中的作用

基因嵌合现象普遍存在于各种癌症中,嵌合基因（chimeric gene）和蛋白在肿瘤发生中发挥重要作用。嵌合基因在人类多种疾病,特别是癌症中过度表达,但分子机制并不明确。近年来,有关嵌合基因的分子机制逐渐清晰,人们对发病较高的前列腺癌、肺癌以及白血病中过度表达的嵌合基因进行深入探索,也为未来肿瘤的诊断和治疗提供非常重要的理论依据。     

刺苋花粉泛过敏原Profilin的抗原性评估与三级结构分析

目的 克隆刺苋花粉中Profilin蛋白基因,分析同源序列中不同性质氨基酸对抗原性及j级结构的贡献.方法 基于生物信息学分析已知泛过敏原Profilin的氨基酸序列并获得核心代表序列,以之为基础设计合成引物,采用Touchdown PCR技术从刺苋花粉的cDNA池中进行基因克隆,并经菌落PCR和双酶切验证;采用生物信息学软件MULTIPRED及SWISS-MODEL在线软件对所得基因编码蛋白进行抗原性评估及三级结构模拟.结果 从刺苋中获得两个序列不同的泛过敏原基因,分别命名为PRF7和PRF23.蛋白质三级结构显示:PRF7与已知的泛过敏原Profilin存在少数氨基酸的筹异,其空间结构与抗原性没有明显变化;而PRF23与北方豚草花粉泛过敏原Q64LH0之间相似程度较低,而空间结构也呈现明显的筹异;尽管Q64LH0与PRF23的伞序列抗原性均值差异无统计学意义,受一些区段上不同性质的氨基酸改变的影响,PRF23在这些区段上的抗原性显著低于Q64L-HO的抗原性.结论 基于Q64LHO和PRF23同源氨基酸序列的抗原性评估及三级结构分析,获得了不同性质氨基酸对抗原性及三级结构的贡献等信息,映射了南方花粉过敏症发牛率较低的原因所在,也为过敏原遗传改良过程中进行氨基酸置换指明了方向.     

Mtb8.4/hIL12嵌合基因疫苗免疫原性

目的:构建克隆结核分枝杆菌Mtb8.4/hIL12嵌合基因疫苗,并在COS7细胞中表达,研究该疫苗的免疫原性。方法:克隆Mtb8.4/hIL12嵌合基因,并导入真核表达载体pCIneo中,构建pCIneoMtb8.4/hIL12重组真核质粒。用限制性内切酶消化、PCR及DNA序列测定等鉴定后,转染COS7细胞,用RTPCR和Westernblot鉴定Mtb8.4/hIL12嵌合基因在转录水平的表达。用Mtb8.4/hIL12嵌合基因疫苗免疫C57BL/6N小鼠,收集脾细胞培养上清检测细胞因子的水平,并用乳酸脱氢酶(LDH)释放法测定细胞毒性T细胞(CTL)的杀伤作用。结果:pCIneoMtb8.4/hIL12重组质粒构建成功。以其转染COS7细胞后,Mtb8.4/hIL12嵌合基因在转录水平获得表达。Mtb8.4/hIL12嵌合基因疫苗能诱导较强的抗原特异性Th1型细胞免疫应答,IFNγ和IL2的分泌增加,IL4的分泌减少,特异性CTL的杀伤活性增强。结论:成功地构建pCIneoMtb8.4/hIL12重组质粒,并在COS7细胞中表达。构建的Mtb8.4/hIL12基因疫苗具有较强的免疫原性,可明显诱导CTL的杀伤活性。     

幽门螺杆菌oipA基因片段的克隆表达及抗原性分析

目的:分片段克隆表达幽门螺杆菌oipA基因,确定抗原性最强的重组蛋白肽段.方法:从Hp国际标准株NCTC11637中获取oipA全长基因,克隆至pGEM-T载体并鉴定正确,以重组pGEM-T/oipA为模板PCR扩增6个不同大小的oipA基因片段,与表达载体pET-42a连接,转化大肠杆菌BL-21,重组子经PCR、酶切、测序鉴定.IPTG诱导重组蛋白肽段的表达,经Western Breeze 化学发光法鉴定融合蛋白;优化诱导时间和IPTG浓度,诱导重组蛋白大量表达;Ni-NTA His*Bind树脂亲和纯化表达产物;羊抗Hp多克隆抗体,Western blot法检测纯化蛋白的抗原性,间接ELISA法筛选抗原性最强的重组肽段.结果:6条oipA基因片段在大肠杆菌中都得到了表达,表达的蛋白肽段均可被抗Hp多克隆抗体识别,具有抗原性,其中反应性最强的是最小的蛋白肽段.结论:oipA基因片段可在原核系统中表达,所获取的最小的肽段抗原性最强,可望制备检测血清相应抗体的试剂盒.     

汉滩病毒G1S0.7嵌合基因重组腺病毒的构建及鉴定

目的在本室前期工作的基础上构建汉滩病毒M基因G1片段与S基因0.7kb片段嵌合基因的重组腺病毒。方法构建含有汉滩病毒G1S0.7嵌合基因的转移载体pShuttle-G1S0.7,然后通过特异性的酶切将嵌合基因与腺病毒DNA相连,电转化E.coli JM109,获得重组腺病毒Adeno-G1S0.7 DNA,转染HFX293细胞得到重组腺病毒。进一步对重组腺病毒的滴度和表达产物进行鉴定。结果构建的含G1S0.7嵌合基因的重组腺病毒,滴度可达10^13～10^15 PFU／L;该重组腺病毒感染Vero-E6细胞后,表达出可被抗汉滩病毒核蛋白及糖蛋白G1的特异性单抗（mAb）所识别的融合蛋白。结论利用腺病毒表达系统,成功地表达同时具有核蛋白及糖蛋白G1生物学活性的融合蛋白,为进一步研究其免疫学特性奠定了基础。     

抗癌胚抗原单克隆抗体可变区基因的克隆及鼠-人嵌合抗体基因在E.coli中的高效表达

本文采用反转录-聚合酶链反应（RT-PCR）技术,从抗癌胚抗原（CEA）单克隆抗体杂交瘤细胞中克隆到了该抗体的重、轻链可变区（Ⅴ区）基因,并分别将其与人的恒定区基因 C_(3γ)、C_κ相拼接,构建鼠-人嵌合抗体基因。SDS-PAGE和Western-Blot分析结果证实嵌合重、轻链抗体基因在E.coli中得到高效表达,表达量约为菌体可溶蛋白的30％和15％。放射免疫分析法（RIA）和间接ELISA测定的结果表明嵌合重链基因表达产物具有与CEA结合的能力。     

2004-2005年中国A(H1N1)亚型流感病毒抗原性及基因特性研究

目的 阐明2004-2005年中国流行的A（H1N1）亚型流感病毒血凝素抗原性及其基因变异情况.方法 对2004-2005年分离的A（H1N1）亚型毒株先进行单向血凝抑制试验;在此基础上选取不同时间、地点的A（H1N1）亚型流感毒株进行血凝素基因HA1区核苷酸序列测定并推导出其氨基酸序列,然后进行基因进化特性分析.结果 单向血凝抑制实验结果表明,2004年A（H1N1）亚型病毒株对鉴定血清的血凝抑制效价与A/Shanghai/1/1999（H1N1）毒株没有4倍差异;2005年分离的A（H1H1）亚型毒株中有62株（占6.2%）病毒与A/Shanghai/1/1999（H1N1）毒株本身的血凝抑制效价相比有4倍差异.HA1区核苷酸序列和氨基酸序列分析表明,我国2005年分离到的A（H1N1）亚型流感病毒株有以下位点发生变异,54 K＞R、90T＞K、101Y＞H、149R＞K、169V＞A、190D＞N、212R＞K、219K＞R、245W＞R、246Y＞F、258T＞N、318V＞A,其中54、190位氨基酸位于抗原决定簇.结论 我国2005年分离的A（H1N1）亚型流感毒株基因特性和抗原性已开始发生变异.     

抗人CD25嵌合抗体基因的构建及其瞬时表达研究

目的:构建抗人CD25嵌合抗体基因并在哺乳动物细胞中进行瞬时表达和初步鉴定。方法:采用RLM-RACE法克隆WuTac抗体可变区和信号肽序列,并利用基因拼接法构建嵌合抗体基因。用脂质体法瞬时转染三种哺乳动物细胞,并使用ELISA、FCM、WB、Dot blot和免疫荧光法进行检测。结果:成功克隆WuTac抗体可变区和信号肽序列,并构建了抗人CD25嵌合抗体表达质粒。瞬时转染结果表明所表达的嵌合抗体保留了亲本抗体WuTac的抗原结合力。结论:成功构建了抗人CD25嵌合抗体基因,为其进一步研究打下基础。     

Characteristics of five monoclonal antibodies to major allergens of the short ragweed pollen

Monoclonal antibodies against major allergens of the short ragweed pollen were produced by fusion of NS-1 cells with splenic cells from BaLB/C mice that had been immunized separately with the major allergens, AgE-B + E-C and AgK, of the short ragweed pollen. These monoclonal antibodies were detected by enzyme-linked immunosorbent assay and further characterized by immunoblot analysis using the crude extract and highly purified allergens of the ragweed pollen. Three monoclonal antibodies obtained by immunization with AgE-B + E-C, designated as 36-6, 27-2 and 48-5, reacted mainly with the beta (36-6) and alpha (27-2) subunit of AgE and both AgE and AgK (48-5), respectively. Two monoclonal antibodies obtained by immunization with AgK, 4-7 and 8-5, had a similar reactivity with AgE-B + E-C, AgK, AgK-A and AgK-B. In addition, however, antibody 4-7 also reacted with AgE-B2 as well as the 36- and the 24- to 22-kilodalton antigens of the crude extract. All 5 monoclonal antibodies were characterized as IgGl subclass. Besides, monoclonal antibody 48-5 also showed cross-reactivity to components of sage pollen. Beyond academic interests, the monoclonal antibodies described here may also be useful in clinical allergy.     

Array-based profiling of ragweed and mugwort pollen allergens

Background: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. Objective: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme‐linked immunosorbent assay (ELISA). Methods: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. Results: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen‐allergic patients. Extensive cross‐reactivity was observed for both patient groups mainly involving the pan‐allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. Conclusions: Molecule‐based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.     

Rapidly released allergens from short ragweed pollen: II. Identification and partial purification

We have identified several basic allergens in 16-min extracts of short ragweed (Ambrosia elatior) pollen and obtained a partially purified fraction (G50 II) using a combination of ion-exchange (CM-Sephadex C25) and gel-filtration (Biogel P30 and Sephadex G50) procedures. G50 II was allergenically and antigenically distinct from known allergens antigen E, Ra3, and Ra5. It gave a highly symmetrical peak on Sephadex G50 (fine), corresponding to a molecular weight of 11,500 daltons, and a single band on SDS polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. However, two cathodic antigens were detectable by both agarose electrophoresis and crossed immunoelectrophoresis. Allergenic activity of G50 II, assessed by puncture testing in ragweed-sensitive subjects, showed a characteristic pattern of response independent of patient response to AgE, Ra3, and Ra5. The major antigenic and allergenic component of G50 II is designated Ra6.     

The spectrum of allergens in ragweed and mugwort pollen

Ragweed and mugwort are important allergenic weeds belonging to the Asteraceae or Compositae plant family. Pollen of mugwort is one of the main causes of allergic reactions in late summer and autumn in Europe and affects about 10-14% of the patients suffering from pollinosis. Ragweed pollen represents the major source of allergenic protein in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is now rapidly increasing particularly in certain areas in France, Italy, Austria, Hungary, Croatia, and Bulgaria. Amb a 1 and Art v 1, the major allergens of ragweed and mugwort, respectively, are unrelated proteins. Amb a 1 is an acidic 38-kDa nonglycosylated protein. The natural protein undergoes proteolysis during purification and is cleaved into a 26-kDa alpha chain, which associates noncovalently with the beta chain of 12 kDa. The two-chain form seems to be immunologically indistinguishable from the full-length molecule. Art v 1 is a basic glycoprotein comprising two domains: an N-terminal cysteine-rich, defensin-like domain and a C-terminal proline/hydroxyproline-rich module. The proline/hydroxyproline-rich domain was recently shown to contain two types of glycosylation: (1) a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core substituted by a variable number (5-28) of alpha-arabinofuranose residues forming branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses, and (2) single and adjacent beta-arabinofuranoses linked to hydroxyproline. As described for other pollen, ragweed and mugwort pollen also contain the pan-allergen profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.     

The heat stability of short ragweed pollen extract and the importance of individual allergens in skin reactivity

A short ragweed pollen extract that was heated in a boiling-water bath remained skin reactive for some allergic individuals and lost reactivity for others. Comparison of heated and unheated extract by immunodiffusion showed that AgE, AgK, and Ra3 could not be detected in the boiled extract but Ra5 was present. By inhibition of the radioallergosorbent test the concentration of Ra5 remained essentially unchanged, but the concentrations of AgE and Ra3 were greatly diminished. The sera of these ragweed-allergic individuals were analyzed for IgE antibodies to AgE, Ra3, and Ra5. Skin reactivity in 17 ragweed-allergic individuals to the heated extract correlated significantly with the levels of IgE antibodies to Ra5. However, the large number of individuals reacting to the boiled extract indicates that Ra5 is not likely to be the only heat-stable allergen. Skin reactivity of the unheated extract did not correlate with IgE antibody levels to any allergen. The results underscore the importance of individual allergens in the materials used for skin testing.     

Rapidly released allergens from short ragweed pollen: I. Kinetics of release of known allergens in relation to biologic activity

Study of the kinetics of in vitro release of known antigens from short ragweed pollen revealed a slow release of AgE (1.5% to 4% release after 16 min) but a rapid release of other highly allergenic components. The rapidly released allergens in 16-min pollen extracts were found to be mainly highly basic proteins including the well-characterized Ra5 molecule along with several hitherto uncharacterized components. Considering previous data that suggest that particles of a similar size to ragweed pollen grains remain in the nose only about 6 to 8 min following inhalation, it is difficult to explain the apparent anomaly between the high biologic potency of 16-min extracts and their low AgE content. Increase in the pH of the extracting buffer from the physiologically normal nasal pH of 5.5 to 6.5 to about pH 8.0 to 8.5 (characteristically found in rhinitis) increased the proportion of AgE released after 16-min extraction by about 10-fold, suggesting that response to allergens in ragweed and other inhalants may enhance AgE release and thereby aggravate allergic symptoms. Detailed comparison of the allergenic activity of AgE and of 16-min and 4-day ragweed extracts in 38 ragweed-sensitive subjects suggested that allergens other than AgE were together more important than AgE in causing ragweed allergy in most patients, although large patient-to-patient differences in relative response were observed. Our data emphasize the need to reevaluate materials used in both diagnosis and treatment of ragweed allergy. The importance of AgE relative to other ragweed pollen components with respect to induction of allergic symptomatology deserves to be placed in a more balanced perspective.     

Airborne ragweed allergens: association with various particle sizes and short ragweed plant parts

These investigations were undertaken to size airborne particles by use of a high-volume sampler and to measure the short ragweed allergen activity of airborne particles of different sizes. We found both in vitro and in vivo short ragweed allergen activity in particles of all size ranges including greater than 6, 3 to 6, 1.8 to 3, 1 to 1.8, and 0.3 to 1.0 micron in diameter. Furthermore, we investigated various parts of the short ragweed plant as possible sources of allergen. Plant parts collected before, during, and after the pollination season demonstrated significant in vitro and in vivo allergen activities. We demonstrated allergen activity in various plant parts, especially the inflorescence, as late as November 30. Appreciable ragweed allergenic activity was also associated with particles less than 1 micron in diameter. Collectively, these observations suggest persistent ragweed plant debris in different sized particles as a source of allergen in the air before and after the ragweed pollination season. This may contribute to out-of-season symptoms observed in highly ragweed-sensitive individuals.     

豚草花粉泛过敏原同源基因克隆与序列分析

目的　克隆豚草花粉泛过敏原同源基因。方法　采用生物信息学分析方法对众多的花粉过敏原基因进行序列同源性比较 ,以序列保守区域为依据设计合成简并引物 ,在特殊的RT PCR条件下 ,结合RACE技术对豚草花粉中的过敏原全长基因进行克隆 ;通过Northern杂交及序列分析初步确定基因产物是否为花粉过敏原。结果　获得了 3个新的全长基因。序列分析显示 :所得过敏原同源基因与数十种不同种属来源的过敏原肌动蛋白结合蛋白 (profilin)具有较高的同源性 ,初步认定其为泛过敏原 ,并暂命名为Amba 8(t)。Northern杂交证实该基因在花粉中表达。结论　采用本研究方法成功地在豚草花粉中克隆到 3个泛过敏原基因 ,为豚草花粉重组过敏原的蛋白质表达及标准化奠定了物质基础     

The characterization of some of the antigens and allergens in ragweed pollen.

Ragweed extract (RW) and antigens E, K, BPA-R and Ra3 were characterized by electrophoresis on starch and polyacrylamide gels. RW extract and antigen E were also subjected to crossed immunoelectrophoresis. All the antigens contained isomers and contaminating proteins. RW separated by starch gel electrophoresis was skin tested and allergens unrelated to antigens E, BPA-R, Ra3 and K were found.     

抗人黑色素瘤嵌合抗体真核表达载体的构建与表达

目的：构建嵌合型抗人黑色素瘤抗体的真核表达载体，并实现真核表达。方法：从3株杂交瘤细胞中克隆得到鼠源单抗的可变区基因，插入含有抗人Tac抗原信号肽以及人免疫球蛋白κ轻链恒定区基因和γ1重链恒定区基因的真核表达载体pMH-CA中，转染293T细胞，进行嵌合抗体表达，并用RT-PCR、ELISA、Westem blot免疫印迹等对抗体表达鉴定。结果：成功构建了抗人黑色素瘤人，鼠嵌合抗体，并实现其真核表达。RT-PCR证实了该抗体在mRNA水平上的表达，ELISA和Westemblot证实了抗人黑色素瘤人，鼠嵌合抗体的表达。结论：成功表达了抗人黑色素瘤人-鼠嵌合抗体。