霍乱毒素B亚单位与p277融合基因在大肠杆菌中表达及免疫分析

为了增强多肽表位的免疫原性,构建了霍乱毒素B亚单位(Cholera toxin B subunit,CTB)和p277的融合基因CTB-p277,将该融合基因克隆到pET28a( )中,获得原核表达载体pET28a-CTB-p277;并将该质粒转入大肠杆菌菌株BL21(DE3)中;重组菌株经2%的乳糖诱导5 h后的表达产物用12%的SDS-PAGE分析表明可以表达融合蛋白,融合蛋白占全菌蛋白约40%,且主要以包含体的形式存在。包含体经洗涤、裂解,用DEAE纤维素离子交换纯化,可以获得99.1%的重组蛋白。重组蛋白的包含体经复性后,在体外可以自组装成五聚体。重组蛋白免疫KK-Ay小鼠后,ELISA的分析表明可以产生抗p277的抗体。血糖和体重的测定结果显示,给药组小鼠的血糖有明显的降低,体重也有相应的减轻。同时GM1-ELISA的检测表明,CTB-p277在体外可以和神经节苷脂GM1(Monosialoganglioside)特异结合,具有生物活性。     

重组霍乱毒素B亚单位作为佐剂与DTP共同鼻腔免疫能提高白喉和破伤风抗毒素水平

霍乱毒素B亚单位（CTB）是一种五聚体，能与细胞膜上的GM1神经节苷脂结合。日本名古屋城市大学Isaka等将携带pNU212-CTB的短芽孢杆菌HPD31株表达的重组（r）CTB作为黏膜佐剂，与DTP一同鼻腔免疫，研究rCTB的黏膜免疫佐剂作用。     

产生霍乱毒素B亚单位的重组卡介苗诱导的免疫应答[英]

经粘膜途径接种的抗原的免疫原性通常弱于全身途径接种，因此粘膜抗原接种后需要粘膜佐剂以获得有效水平的免疫应答。已知五聚体形式的霍乱毒素B亚单位(CTB)是有效的粘膜佐剂，当经粘膜途径与抗原一起给予时能增强抗原特异性分泌性IgA     

CTB-Aβ42融合蛋白的原核表达条件优化及鉴定

目的探索融合基因CTB-Aβ42在大肠杆菌中表达的最适条件,并纯化得融合蛋白。方法设置不同的诱导温度、诱导时间、异丙基-β-D-硫代半乳糖苷(IPTG)浓度,在大肠杆菌中表达融合基因CTB-Aβ42,SDS-PAGE凝胶电泳鉴定表达产物,GST亲和色谱柱纯化目的蛋白,Western blot鉴定目的蛋白免疫原性。结果 SDS-PAGE结果显示,GST-CTB-Aβ42融合蛋白分子质量约为45 kD,与预期结果一致;Western blot结果表明,纯化得到的目的蛋白具有免疫原性。在诱导条件为30℃,0.1 mmol/L IPTG诱导2 h表达的可溶性蛋白所占比例最高;在37℃,0.1mmol/L IPTG诱导4 h表达的融合蛋白总量最高,小部分可溶性表达,大部分以包涵体的形式存在。结论本实验对CTB-Aβ42融合蛋白的原核表达条件进行了优化,经GST亲和色谱柱纯化获得具有免疫原性的融合蛋白。     

rhCG融合蛋白在CHO细胞中的表达、纯化与鉴定

为实现rhCG融合蛋白在CHO细胞中的高效表达,构建两个不同的 His－tag rhCG 融合蛋白真核表达载体,通过瞬时转染比较两个载体在CHO－dhfr－细胞中的表达量,选择表达量较高的载体稳定转染CHO－dhfr －,并采用氨甲喋呤（ MTX）进行加压筛选高表达单克隆细胞株。加压后,表达量由1日1300mIU? mL －1提高至18000mIU? mL －1（以106个细胞计）。该克隆株经传代50代以后,rhCG表达量未发生明显变化,表明该克隆为稳定克隆株。用Ni＋螯合亲和层析及凝胶过滤层析分离纯化表达产物,获得纯度大于99％的rhCG融合蛋白,SDS－PAGE电泳及 Western Blot结果表明rhCG得到正确及完整的表达,生物学活性测定结果表明样品比活为11000IU? mg －1。     

Non-ionic surfactant vesicles mediated transcutaneous immunization against hepatitis B

Transcutaneous immunization (TI) has many practical merits compared to parenteral routes of administration. In the present study, non ionic surfactant vesicular carrier, i.e. niosomes, was evaluated for topical delivery of vaccines using hepatitis B surface protein as an antigen and cholera toxin B as an adjuvant. Niosomes were characterized for size, shape, entrapment efficiency and in process antigen stability. In vitro permeation and skin deposition studies of antigen were performed using human cadaver skin. Skin penetration efficiency of niosomes was assessed by confocal laser scanning microscopy. The immune stimulating activity of these vesicles was studied by measuring the serum IgG titer, isotype ratio IgG2a/IgG1and mucosal immune responses following transcutaneous immunization in Balb/c mice and results were compared with the alum adsorbed HBsAg given intramuscularly and topically administered plain HBsAg solution. The result shows that optimal niosomal formulation could entrap 58.11 ± 0.71 of antigen with vesicle size range of 2.83 ± 0.29 μm. Serum IgG titers after three consecutive topical administrations were significantly better than single administration of hepatitis antigen with niosomal system, suggesting an effective stimulation of serum immune response; higher IgG1/IgG2a ratio revealed CTB mixed niosomes elicit both Th1 and Th2 responses. This study suggests that topical immunization with cholera toxin B is potential adjuvant for cutaneous immune responses when coadministered with the HBsAg encapsulated niosomes. Results also suggest that the investigated niosomes systems can be effective as topical delivery of vaccines.     

可承载CETP多肽表位的颗粒样蛋白表达载体的克隆、表达与分离纯化

为了提高多肽表位的免疫原性，利用乙肝病毒核心抗原能够在体外自行组装成病毒样颗粒的特性，将人源胆固醇酯转运蛋白羧基端26个氨基酸连同大肠杆菌DnaK基因C端结构域克隆人乙肝病毒核心抗原的免疫优势决定区，并在大肠杆菌BL-21（DE3）表达，以包含体形式存在，经过洗涤、复性和分子筛纯化，获得纯度达90％以上的目的蛋白。纯化的蛋白免疫新西兰大白兔后能够诱导产生高水平的抗胆固醇酯转运蛋白的抗体，表明乙肝病毒核心抗原能够在体外自发装配成多聚体颗粒，并能够将插入其免疫优势决定区的胆固醇酯转运蛋白（CETP）多肽表位展示于颗粒表面以提高免疫原性，显示出其作为疫苗免疫载体的良好前景。     

Uptake and intracellular fate of cholera toxin subunit b-modified mesoporous silica nanoparticle-supported lipid bilayers (aka protocells) in motoneurons

Cholera toxin B (CTB) modified mesoporous silica nanoparticle supported lipid bilayers (CTB-protocells) are a promising, customizable approach for targeting therapeutic cargo to motoneurons. In the present study, the endocytic mechanism and intracellular fate of CTB-protocells in motoneurons were examined to provide information for the development of therapeutic application and cargo delivery. Pharmacological inhibitors elucidated CTB-protocells endocytosis to be dependent on the integrity of lipid rafts and macropinocytosis. Using immunofluorescence techniques, live confocal and transmission electron microscopy, CTB-protocells were primarily found in the cytosol, membrane lipid domains and Golgi. There was no difference in the amount of motoneuron activity dependent uptake of CTB-protocells in neuromuscular junctions, consistent with clathrin activation at the axon terminals during low frequency activity. In conclusion, CTB-protocells uptake is mediated principally by lipid rafts and macropinocytosis. Once internalized, CTB-protocells escape lysosomal degradation, and engage biological pathways that are not readily accessible by untargeted delivery methods.     

抗菌肽Cecropin B-肿瘤血管生长抑制因子 Kringle5融合蛋白表达载体的构建及其表达

通过重叠区扩增法人工合成抗菌肽Cecropin B(CB)基因,从ThioA/L15-7上卸下肿瘤血管生长抑制因子Kringle5(K5)基因,将两者按正确的阅读框架融合并定向克隆至大肠杆菌高效表达载体pET32a上,构建成pET32a/CB-K5重组载体。重组载体转化BL21(DE3)细胞后,提取质粒进行PCR鉴定和序列分析,证明重组质粒pET32a/CB-K5阅读框架和融合基因序列均与预期相符。实验结果显示,成功地构建了抗菌肽CecropinB(CB)和肿瘤血管生长抑制因子Kringle5(K5)融合蛋白的表达载体pET32a/CB-K5。转化E.coliBL21(DE3),SDS-PAGE分析显示,在IPTG诱导下,融合蛋白以包涵体的形式在重组转化菌株中获得高效表达,表达量达到菌体总蛋白的50%。     

DNA聚合酶β在宫颈上皮内瘤变和宫颈癌中的表达及临床意义

目的 探讨DNA聚合酶β(DNA polymerase beta,polβ)在宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)和宫颈癌(cervical carcinoma,CC)中的表达及其临床意义.方法 采用RT-PCR方法检测正常宫颈、CIN、CC组织中polβ mRNA的表达;Western Blot检测这些组织中polβ蛋白相对表达量.结果 正常宫颈、CIN Ⅰ、CINⅡ、CINⅢ、CC组织polβ mRNA分别为(0.23±0.08)、(0.25 ±0.11)、(0.36±0.18)、(0.62±0.22)、(0.85±0.28);正常宫颈、CINⅡ、CINⅢ、CC组间polβ mRNA表达差异有统计学意义(P＜0.05).正常宫颈、CIN Ⅰ、CINⅡ、CINⅢ、CC组织polβ蛋白相对表达量分别为(19±8)％、(18±6)％、(43±17)％、(72±26)％、(93±31)％;正常宫颈、CINⅡ、CINⅢ、CC组间pol β蛋白表达差异有统计学意义(P＜0.05).CIN Ⅰ组织polβ mRNA和蛋白表达与正常宫颈组织比较差异均无统计学意义(P＞0.05).结论 从CINⅡ至CCpolβ表达逐步递增,pol β在CC发生发展中起重要作用,polβ可能成为预测CC进展的生物学指标.     

宫颈鳞癌组织中组织蛋白酶B、层粘连蛋白表达及其意义

目的探讨宫颈鳞癌组织中组织蛋白酶B（Cathepsin B,CB）、层粘连蛋白（Laminin,LN）表达情况,研究其与宫颈鳞癌浸润及转移的内在关系。方法采用免疫组化SP法检测60例宫颈鳞癌组织中CB、层粘连蛋白的表达。结果CB高表达与宫颈鳞癌淋巴结转移、浸润深度关系密切（P〈0.05）;LN表达下调与宫颈鳞癌淋巴结转移、浸润深度关系密切（P〈0.05）;宫颈鳞癌中CB和LN蛋白表达呈负相关（P〈0.05）。结论宫颈鳞癌组织中LN阳性表达率下降,CB阳性表达率升高,两者在宫颈鳞癌的浸润转移过程中起着重要作用;联合检测上述两种指标的表达情况将有助于宫颈鳞癌的早期诊断和预后判断。