Drawn gelatin films with improved mechanical properties

Chain anisotropic distribution in gelatin films has been obtained by uniaxial stretching at constant relative humidity, followed by air drying and successive cross-linking with glutaraldehyde. The drawn samples have been characterized by mechanical tests, differential scanning calorimetry and scanning electron microscopy. The Young’s modulus, E, and the stress at break, σ_b, increase linearly with the draw ratio and reach values which are about five times those characteristic of undrawn samples. Furthermore, on stretching the alignment of the gelatin strands along the direction of deformation increases while the thickness of the layers decreases significantly. The renaturation level, that is the fraction of gelatin in a collagen-like structure, has been calculated as the ratio between the melting enthalpy of gelatin samples and that of tendon collagen. The results indicate that the improvement of mechanical properties achieved by drawn gelatin is closely related to the renaturation level. The experimental approach utilized to induce segmental orientation in gelatin films, allows to obtain anisotropic materials with improved mechanical properties in the direction of deformation, and can be usefully applied in the preparation of biomaterials.     

瑞替普酶融合蛋白在大肠杆菌中表达条件的优化及其复性

目的:为优化瑞替普酶(reteplase,r-PA)在大肠杆菌中的高效融合表达的条件,并提高其复性率.方法:通过改变诱导时间和温度、诱导剂浓度、培养基pH值及氨苄青霉素(Amp)浓度等条件,利用SDS-PAGE分析以上条件的改变对表达产物产量的影响.运用金属螯合层析对融合蛋白进行纯化和复性.结果与结论:LB培养基pH值为6,Amp浓度为100μg/mL,乳糖浓度为5 mmol/L的条件下39℃诱导4 h可获得高效表达的r-PA融合蛋白,其表达量占全菌蛋白的70%,其产物主要以包涵体形式存在.融合蛋白运用金属螯合层析一步纯化复性,其复性率可达10%,比活为55.7 U/μL.     

Expression,production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10⁻⁸ M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10⁻⁷ M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.     

抗氯霉素单链抗体的高效生产

[目的]建立抗氯霉素单链抗体(scFvCAP)的大肠杆菌高效生产体系。[方法]采用重叠延伸PCR法组装scFvCAP,并通过优化5’端序列实现高效表达,采用β-环糊精人工伴侣系统实现SDS变性蛋白的复性。[结果]成功组装scFvCAP基因(Genebank ID:GU258048)。在强启动子T7驱动下,scFvCAP基因独立表达时未见显著表达,而通过同义突变所得突变基因scFvCAP mut(Genebank ID:GU258048)即可高效表达。高效表达所形成的包涵体蛋白可溶于1%SDS-8mol/L尿素溶液,经β-环糊精人工伴侣系统复性后所获scFvCAP具有与母本单克隆抗体相似的亲和力。[结论]成功建立了基于大肠杆菌基因工程系统的scFvCAP高效生产体系。     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

抗人肝癌基因工程嵌合Fab抗体的原核表达及复性

目的　分别在大肠杆菌中高效表达嵌合Fd及嵌合轻链 ,并进行嵌合Fab复性的研究。方法　分别将嵌合Fd及嵌合轻链基因插入到原核表达载体pET32a中 ,构建成重组载体pET32a/cFd和pET32a/cL。转化大肠杆菌后诱导其表达。大量制备表达的嵌合Fd及嵌合轻链后 ,按二者绝对量等摩尔比混合后进行透析复性。然后 ,分别利用SDS PAGE、Westernblot和ELISA进行分析和鉴定。结果　成功获得了嵌合Fd及嵌合轻链的原核非融合高效表达 ,表达蛋白相对分子质量 (Mr)均在 2 4×10 3 左右。表达量分别约占菌体总蛋白的 2 8.3%和 32 .3% ,2种目的蛋白均主要以不溶性的包涵体形式存在。另外 ,嵌合Fd与嵌合轻链在缓慢透析的条件下成功地复性为嵌合Fab抗体 ,Mr 约为 4 2×10 3 ,具有特异性抗原结合活性和良好的亲和力。在起始总蛋白浓度为 10 0 μg/ml时 ,复性效率约为4 9 %。结论　成功实现了嵌合Fab抗体的高效表达及高效复性 ,为进一步探讨其在肝癌治疗中的应用奠定了基础     

结核分枝杆菌分泌蛋白MPT 64的克隆、表达、纯化和复性

目的构建结核分枝杆菌分泌蛋白MPT 64的重组质粒,并在大肠杆菌中表达及纯化和复性重组蛋白。方法用PCR方法从结核分枝杆菌H37Rv基因组中扩增出mpt 64基因片段,插入PKRX-T载体中,再亚克降到表达载体PET30a中,在大肠杆菌中表达;应用组氨酸标签(His-Tag)纯化重组蛋白;在PBS中通过半透膜恢复重组蛋白的天然结构。结果构建了含MPT 64重组质粒的大肠杆菌工程菌,并以包涵体形式表达重组蛋白MPT 64,其占细胞总蛋白的20%~30%;重组蛋白变性后经镍柱纯化,其纯度达90%以上;通过半透膜完全恢复重组蛋白的天然结构。结论具有天然结构和高纯度的重组融合蛋白有可能成为有效的结核病血清学诊断的候选抗原。     

蓝藻抗病毒蛋白N基因的原核表达及重组蛋白的纯化和复性

目的:构建蓝藻抗病毒蛋白(CV-N)基因原核表达载体,表达、纯化并复性其重组蛋白。方法:根据GenBank中CV-N的全基因序列,人工合成CV-N序列,进行PCR扩增。将扩增的PCR产物克隆至原核表达载体pET30a( )上,构建重组表达载体pET30a-CV-N,测序鉴定后,转化E.coliBL21(DE3),IPTG诱导表达蛋白,SDS-PAGE和Westenblot鉴定表达产物,Ni Sepharose柱纯化目的蛋白,稀释法复性蛋白。结果:重组质粒pET30a-CV-N测序结果显示,插入片段为303 bp,与GenBank中的CV-N基因序列完全相同。经IPTG诱导的CV-N基因可在E.coli中高效表达;SDS-PAGE分析表明,相对分子质量11000处出现一条新条带,主要以包涵体形式存在,37℃诱导2、4、6、8 h后,表达蛋白分别占菌体蛋白的3.87%、19.10%、33.98%和31.58%。Western blot结果显示,重组蛋白能与抗His单抗特异性反应。将诱导6 h的菌体超声破碎后,Ni Sepharose柱纯化,相对分子质量11 000处显示出清晰的单一条带,且蛋白复性效果良好。结论:成功地构建了CV-N原核表达载体,表达并纯化出重组蛋白,为深入研究其抗病毒的活性奠定了基础。     

Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140

Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC₅₀ values between 1.0 and 6 μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI₅₀ values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9–Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.     

猪干扰素-γ基因表达产物的纯化与复性研究

目的：纯化、复性猪干扰素-γ基因（poIFN-γ）表达产物并分析其抗病毒活性。方法：用异丙基-β—D-硫代半乳糖苷（Isopropylthio—β—D—galactoside,IPTG）诱导原核表达载体pQE30／poIFN-γ,超声裂解包涵体,尿素溶解、Ni—NTA纯化和透析复性后,对所得的蛋白进行抗病毒活性检测。结果：经复性纯化的目的蛋白纯度达95％以上,在PK15细胞上抗滤泡性口炎病毒比活性为6．2×10^5u／mg。结论：获得PoIFN-γ活性蛋白,为进一步研究应用奠定基础。