Anti-HER-2-SEC2融合免疫毒素的构建和活性研究

以PCR技术从金黄色葡萄球菌基因组DNA中首次克隆编码成熟SECZ蛋白的全基因sec2。该基因共717bp，编码239个氨基酸，Genbank Accession number：AY450554。构建了SEC2的表达载体pET-28a-sec2，并在大肠杆菌BL21（DE3）中高效表达可溶性rSEC2蛋白。经亲和层析纯化，其纯度在95％以上，平均回收量为每升培养物40mg。纯化的rSEcZ保持了与野生型相当的生物学活性。以限制性核酸内切酶连接技术分别将两个抗人表皮生长因子受体HER-2单链抗体基因通过DNA Linker与sec2融合，构建融合基因b-l-sec2和ml小sec2，并以两种方式表达纯化。以pET-32a表达载体在E，coliAD494（DE3）中以氨基端融合大肠杆菌硫氧还蛋白（TrxA）形式高效表达融合蛋白TRX-B-L-SEC2和TRX-ML-L-SEC2，经亲和层析纯化，并以肠激酶切割得到成熟融合免疫毒素B-L-SEC2和ML-L-SEC2，其纯度在95％以上，平均回收量为每升培养物smg；以构建的新型表达载体pASK-75-EX在E.coliBL21（ED3）中以不溶性包涵体形式表达融合免疫毒素蛋白，经变性、纯化和复性后得到具有生物学活性的融合免疫毒素，其纯度在95％以上，平均回收量为每升培养物30mg。以两种方式制备的融合免疫毒素都保持了SECZ蛋白的免疫原性，都能有效刺激人外周血单个核细胞的增殖，并且都显示出在体外与HER-2过表达的乳腺癌细胞SK-Br-3特异性结合能力，具有显著的靶向性抑瘤作用。用PcR方法扩增了编码TrxA蛋白的基因trxA并克隆至表达载体pET-28a启动子上游，构建了一种在单质粒中利用两个相同的启动子游离共表达硫氧还蛋白与目的蛋白的表达载体。利用该载体可使TrxA与外源蛋白在大肠杆菌BL21（DE3）中以非融合形式高效共表达。共表达的TrxA可明显促进外源蛋白单链抗体ML3.9（scFv-ML）、3一轻基苯甲酸-6-单加氧酶（3HBA）的可溶性表达；并明显减少肠毒素C2（SEC2）、结核杆菌螺旋酶A亚基（GYRA）的包涵体表达。

Anti-inflammatory and immunomodulatory activities of polysaccharide from Chlorella stigmatophora and Phaeodactylum tricornutum

Crude polysaccharide extracts were obtained from aqueous extracts of the microalgae Chlorella stigmatophora and Phaeodactylum tricornutum. The crude extracts were fractionated by ion-exchange chromatography on DEAE-cellulose columns. The molecular weights of the polysaccharides in each fraction were estimated by gel filtration on Sephacryl columns. The crude polysaccharide extracts of both microalgae showed anti-inflammatory activity in the carrageenan-induced paw edema test. In assays of effects on the delayed hyper-sensitivity response, and on phagocytic activity assayed in vivo and in vitro, the C. stigmatophora extract showed immunosuppressant effects, while the P. tricornutum extract showed immunostimulatory effects. Copyright © 2003 John Wiley & Sons, Ltd.

Multifunctional metallophosphors with anti-triplet–triplet annihilation properties for solution-processable electroluminescent devices

With the goal to provide organometallic triplet emitters with good hole-injection/hole-transporting properties, highly amorphous character for simple solution-processed organic light-emitting diodes, and negligible triplet-triplet (T-T) annihilation, a series of new phosphorescent cyclometalated Ir-III and Pt-II complexes with triphenylamine-anchored fluorenylpyridine dendritic ligands were synthesized and characterized. The photophysical, thermal, electrochemical and electroluminescent properties of these molecules are reported.

Mechanism and life study on polyaniline anti-corrosion coating

Solvent free polyaniline emeraldine base(EB) corrosion protection coating was prepared, employing aliphatic polyamine as solvent of EB as well as hardener of epoxy resin. This coating passed 2000h of salt fog test when the EB loading was about 1 wt%. The interaction between EB and iron indicated that EB acted as a "quasi-catalyst" to cause the formation of densed iron oxide film in the interface.

The chemical resolution of racemic cis-2-hydroxymethyl-5(cytosine-1 '-yl)-1,3-oxathiolane (BCH-189)-one direct method to obtain lamivudine as anti-HIV and anti-HBV agent

Racemic cis-BCH-189 can be resolved to (-)-enantiomer (lamivudine) and (+)-enantiomer by esterification of cis-2-hydroxymethyl-5-(N-4(')-acetylcytosine-1'-yl)-1,3-oxathiolane and (+)-menthyl chloroformate in CH3CN with pyridine as base. The two diastereomers of ester were seperated by recrystallization in methanol at 0degreesC. Lamivudine was obtained by deprotection of (-)-diastereomer with high yield.

Amplified immunoassay of human IgG using real-time biomolecular interaction analysis (BIA) technology

An automated biomolecular interaction analysis instrument (BI-Acore) based on surface plasmon resonance (SPR) has been used to determine human immunoglobulin G (IgG) in real time. Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran modified gold film surface. The samples of human IgG prepared in HBS buffer were poured over the immobilized surface. The signal amplification antibody was applied to amplify the response signal. After each measurement, the surface was regenerated with 0.1 mol/L H3PO4. The assay was rapid, requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding, antibody amplification and regeneration. The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L. The same antibody immobilized surface could be used for more than 110 cycles of binding, amplification and regeneration. The results demonstrate that the sensitivity, specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.

Preparation of rare earth ion induced conformation-specific anti-calmodulin monoclonal antibody

Monoclonal antibody technique was employed to detect the conformational change of calmodulin induced by metal ions. Bovine calmodulin was firstly modified by 2,4-dinitrofluorobenzene to improve its immunogenicity, then, the derived protein was saturated with trivalent europium ions and injected to Balb/c mice as antigen. After four times of immunization, a corresponding antibody was detected and its titer in serum was determined as 1 : 12 000. By fusing of the spleen cells with hybridoma cells, a europium induced conformation-specific anti-calmodulin monoclonal antibody cell strain named as 2C3 was produced successfully. The molecular recognition ability of antibody to apocalmodulin and holocalmodulin showed a significant difference, indicating that this antibody could be applied to the studies of different effects of metal ions on the conformational change of calmodulin and its interaction with target molecules.

Preparation of europium induced conformation-specific anti-calmodulin monoclonal antibody

Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.