Hsp70与汉滩病毒S基因融合蛋白的原核表达及免疫小鼠的初步研究

本文构建了hsp70与S基因的原核融合表达载体pGEX-4T-1/hsp70-S,在大肠杆菌中表达,并通过GSTrapFF柱进行了纯化。同时制备了NP和Hsp70两种纯化蛋白。分别用这三种纯化蛋白免疫BALB/c小鼠,结果表明纯化的NP和Hsp70-NP两种蛋白均可同时诱导产生抗汉滩病毒核蛋白(NP)抗体,且后者刺激产生的抗体效价明显高于前者。淋巴细胞增殖实验表明,两组免疫小鼠的脾细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP组免疫小鼠脾细胞对NP的增殖指数明显高于NP组免疫组。结果显示,与单独用NP免疫小鼠相比,Hsp70-NP纯化蛋白可以刺激机体产生更强的抗汉滩病毒体液免疫应答和特异性淋巴细胞增殖反应。     

Hsp70-NP融合蛋白增强诱导HTNV NP特异性CTL的实验研究

本文采用纯化蛋白Hsp70-NP,NP,Hsp70分别免疫C57/BL6小鼠,取各组小鼠脾淋巴细胞进行淋巴细胞增殖试验和细胞毒试验.此外,为了获得细胞毒实验的靶细胞,本文还采用脂质体介导质粒pcDNA3.1/S转染黑色素瘤细胞B16,通过G418筛选稳定克隆,并用RT-PCR,Western blots以及免疫荧光染色证实N蛋白在胞浆中表达.淋巴细胞增殖实验表明,Hsp70-NP,NP组小鼠脾淋巴细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP组的增殖指数明显高于NP免疫组.细胞毒实验结果表明,LDH的释放具有效应细胞依赖性,Hsp70-NP,NP免疫组脾淋巴细胞均可以特异性杀伤靶细胞B16-N,而Hsp70-NP免疫组的杀伤率显著高于NP免疫组.实验结果显示,Hsp70可以增强NP诱导产生特异性CTL的能力.本研究结果为进一步设计基于NP的合成肽疫苗或基因疫苗提供了重要实验依据.     

Hsp70-NP融合蛋白增强诱导HTNV NP特异性CTL的实验研究

本文采用纯化蛋白Hsp70-NP,NP,Hsp70分别免疫C57/BL6小鼠,取各组小鼠脾淋巴细胞进行淋巴细胞增殖试验和细胞毒试验。此外,为了获得细胞毒实验的靶细胞,本文还采用脂质体介导质粒pcDNA3.1/S转染黑色素瘤细胞B16,通过G418筛选稳定克隆,并用RT-PCR,Westernblots以及免疫荧光染色证实N蛋白在胞浆中表达。淋巴细胞增殖实验表明,Hsp70-NP,NP组小鼠脾淋巴细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP组的增殖指数明显高于NP免疫组。细胞毒实验结果表明,LDH的释放具有效应细胞依赖性,Hsp70-NP,NP免疫组脾淋巴细胞均可以特异性杀伤靶细胞B16-N,而Hsp70-NP免疫组的杀伤率显著高于NP免疫组。实验结果显示,Hsp70可以增强NP诱导产生特异性CTL的能力。本研究结果为进一步设计基于NP的合成肽疫苗或基因疫苗提供了重要实验依据。     

单纯疱疹病毒2gD-Hsp70融合蛋白基因的构建及表达

构建并原核表达Hsp70-HSV2gD融合蛋白。将Hsp70和HSV-2gD蛋白基因分别克隆到原核表达载体pGEX-4T-1,构建成重组质粒pGEX-4T-Hsp70-gD,并测序鉴定。重组质粒pGEX-4T-Hsp70-gD转化大肠杆菌DH5α后,IPTG诱导表达并进行SDS-PAGE分析。表达产物纯化后做Westernblot检测。将其肌注免疫BALB/c小鼠,检测融合蛋白对免疫小鼠脾淋巴细胞增殖、γ-干扰素产生以及血清中gDIgG水平的影响。表达产物的SDS-PAGE分析发现,在相对分子量为118kD处有外源蛋白表达,与预期蛋白带一致。用GST柱得到了纯化的Hsp70-HSV2gD融合蛋白。Westernblot证实,表达产物具有良好的活性。GST-Hsp70-gD组蛋白疫苗免疫的小鼠,其脾淋巴细胞刺激指数和脾淋巴细胞培养上清中γ-干扰素的水平高于其它组(P<0.05)。血清单纯疱疹病毒-2gD蛋白的抗体水平高于其它组(P<0.05)。     

汉滩病毒M和S基因不同拼接方式嵌合基因免疫效果的研究

本文在前期工作的基础上,构建了汉滩病毒76-118株M基因G2片段与S基因5'端0.7Kb片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7及pcDNA3.1-S0.7G2;用该质粒免疫BALB/c小鼠,结果表明两种质粒免疫小鼠可同时诱导产生抗汉滩病毒核蛋白(NP)及糖蛋白(GP)特异性的抗体,且前者刺激产生的抗体效价明显高于后者.淋巴细胞增殖实验表明,pcDNA3.1-G2S0.7组免疫小鼠脾细胞时NP及GP的增殖指数均明显高于空载体对照组,而pcDNA3.1-S0.7G2组未检测到其淋巴细胞有明显的增殖.这说明汉滩病毒M基因G2片段及S基因0.7Kb片段的嵌合基因既可刺激机体产生特异的抗汉滩病毒体液免疫应答,也可刺激机体产生特异的细胞免疫应答.不同拼接方式对嵌合基因免疫效果有很大影响,嵌合基因G2S0.7这种拼接方式明显优于S0.7G2.     

乙脑E蛋白与结核杆菌HSP70的融合蛋白对BALB/c小鼠特异性免疫的影响

研究酵母表达的乙脑病毒(JapaneseEncephalitisvirus)E蛋白主要抗原片段与结核杆菌热休克蛋白70 (hsp70 )形成的融合蛋白对小鼠细胞免疫和体液免疫的影响。采用腹膜内注射蛋白的方法免疫小鼠,以直接免疫E蛋白主要抗原片段和以E蛋白主要抗原片段和单独表达的hsp70两者均以5 0pmol的量进行混合后的蛋白免疫的小鼠作为对照,用半定量RT PCR检测细胞免疫因子IL 2mRNA的水平,IL 2是细胞介导的免疫反应和巨噬细胞激活中的关键分子,MTT法检测淋巴细胞的增殖情况以及通过ELISA检测抗体水平,从这3个方面来评价融合与未融合蛋白以及等量混合后的免疫效果。结果E蛋白主要抗原片段与结核杆菌hsp70重组后,mIL 2 ,淋巴细胞的增殖以及抗体水平均较重组前明显升高,因此,以乙脑E HSP70融合蛋白免疫能增强小鼠针对E蛋白主要抗原片段的细胞免疫和体液免疫     

重组结核分枝杆菌Hsp16.3的纳米金免疫传感器检测Hsp16.3抗体

克隆和表达结核分枝杆菌热休克蛋白16.3(Hsp16.3),建立纳米金免疫传感器检测结核病患者血清Hsp16.3抗体.PCR扩增hsp16.3基因,构建重组表达质粒pQE30-hsp16.3,表达和纯化Hsp16.3,Western blot分析其反应原性;晶种生长法制备金纳米棒并连接Hsp16.3,建立纳米金免疫传感...     

汉滩病毒M和S基因不同拼接方式嵌合基因免疫效果的研究

本文在前期工作的基础上,构建了汉滩病毒76－118株M基因G2片段与S基因5'端0.7Kb片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7及pcDNA3.1-S0．7G2;用该质粒免疫BALB/c小鼠,结果表明两种质粒免疫小鼠可同时诱导产生抗滩滩病核蛋白（NP)及糖蛋白（GP）特异性的抗体,且前者刺激产生的抗体效价明显高于后者。淋巴细胞增殖实验表明,pcDNA3.1-G2S0.7组免疫小鼠脾细胞时NP及GP的增殖指数均明显高于空载体对照组,而pcDNA3.1-S0．7G2组未检测到其淋巴细胞有明显的增殖。这说明汉滩病毒M基因G2片段及S基因0.7Kb片段的嵌合基因既可刺激机体产生特异的抗汉滩病毒体液免疫应答,也可刺激机体产生特异的细胞免疫应答。不同拼接方式对嵌合基因免疫效果有很大影响,嵌合基因G2S0．7这种拼接方式明显优于S0．7G2。     

果蝇Hsp83多克隆抗体的制备及鉴定

目的制备和鉴定抗Hsp83蛋白的多克隆抗体。方法利用PCR技术从果蝇cDNA中获得hsp83基因片段,构建重组质粒;将其转化到BL21(DE3)菌株中诱导蛋白表达,利用Ni-NTA亲和法纯化重组蛋白;再将纯化的蛋白免疫BALB/C小鼠制备多克隆抗体;利用免疫印迹法(Western blot)和免疫荧光染色法检测多克隆抗体的特异性。结果构建的pET28ahsp83质粒在大肠杆菌中成功表达了Hsp83融合蛋白,蛋白纯化后作为抗原免疫小鼠,获得了抗Hsp83的多克隆抗体。免疫印迹法和免疫荧光染色法检测显示,抗果蝇Hsp83多克隆抗体具有较高的特异性,并能检测出内源性Hsp83蛋白。果蝇卵巢免疫荧光染色显示,Hsp83蛋白定位在卵巢细胞的细胞质中。结论成功制备了小鼠抗Hsp83蛋白的特异性抗体,此工作为深入研究Hsp83蛋白的功能奠定了基础。     

棉铃虫Hsp70的多克隆抗体制备及鉴定

HSPs(热休克蛋白)是机体在不利环境条件刺激下合成的一类蛋白质,在进化上高度保守,普遍存在于生物体,其中Hsp70是最为保守的成员。研究发现,昆虫滞育过程中普遍存在Hsp70表达上调的现象。然而,现有的研究均是在基因水平的检测结果。为了能从蛋白水平检测棉铃虫中Hsp70的表达,本研究制备了棉铃虫Hsp70的多克隆抗体。构建了hsp70的原核表达载体并在大肠杆菌BL21(DE3)中成功表达,重组蛋白经镍柱纯化后免疫兔子,制备了棉铃虫Hsp70的多克隆抗体。抗体的效价较高,达到了1∶256 000,此外Western结果表明,制备的抗体能检测出热诱导的Hsp70蛋白。本研究制备了高效价且较为特异的Hsp70对克隆抗体,该抗体为后续从蛋白水平研究棉铃虫滞育过程中Hsp70的表达及其分子机制奠定了基础。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.