重组人干扰素λ1的表达、纯化及生物活性研究

目的:利用大肠杆菌系统表达重组人Ⅲ型干扰素λ1(rhIFN-λ1),并进行纯化,以比较其与重组人干扰素-α2b(rhIFN-α2b)生物学活性的差异。方法:密码子优化的rhIFN-λ1基因与pET-44a载体连接构建原核表达质粒,在大肠杆菌BL21(DE3)中表达,表达产物经系列层析纯化;采用细胞病变抑制法比较纯化的rhIFN-λ1与rhIFN-α2b、市售rhIFN-λ1的抗病毒活性;采用MTS法比较这些干扰素的抗肿瘤细胞增殖活性。结果:基因优化后的rhIFN-λ1在大肠杆菌中得以高效表达,用所建立的纯化工艺可制备得到纯度达95.7%的rhIFN-λ1;rhIFN-λ1的抗病毒比活性为3.2×105 U/mg,比市售rhIFN-λ1的比活性(1.1×105 U/mg)略高,抗肿瘤细胞增殖活性为rhIFN-α2b的1.7倍。结论:获得的rhIFN-λ1具有剂量依赖的抗病毒活性和抗增殖活性,其抗病毒活性比市售rhIFN-λ1略高,抗细胞增殖活性高于rhIFN-α2b,提示rhIFN-λ1有较大的应用前景。     

人干扰素ω(huIFN-ω)的高效表达、纯化及生物学活性

为了获得人干扰素-ω(huIFN-ω)在大肠杆菌中的高效表达,根据大肠杆菌的偏爱密码子,人工设计并合成了huIFN-ω高表达基因,并克隆到原核表达载体pBV220,在大肠杆菌DH5α中实现了高效表达,目的蛋白占细胞总蛋白的15%左右,以包涵体形式存在.表达产物经变性、复性及阳离子和分子筛层析纯化,纯度达90%以上,产物的抗病毒活性约为1.0×108IU/mg,并具有体外促NK杀伤活性.此结果为进一步研究和开发重组huIFN-ω奠定了基础.     

重组胸腺素α1的表达、纯化和生物学活性

为获得重组人胸腺素α1(recombinantthymosinα1,Tα1) ,采用融合表达方式表达Tα1基因 ,重组融合表达载体Tα1 pGEX 4XT 1转化大肠杆菌DE3(lys)构建工程菌 .对工程菌进行补料分批培养并诱导表达 ,得到目的蛋白的可溶性表达 .亲和层析纯化融合蛋白GST Tα1,经凝血酶裂解融合蛋白 ,亲和层析除去GST ,SourceQ离子交换 ,得到Tα1单体 ,得率为 30mg L发酵液 .生物学活性分析显示 ,重组Tα1能显著促进小鼠脾细胞增殖 (P <0 0 1) ,其活性与天然Tα1相似     

重组人TNF-α在大肠杆菌表达、纯化及生物学活性鉴定

[目的]获得人TNF-α蛋白,并鉴定其生物学活性。[方法]通过对人源TNF-α序列进行密码子优化构建了p Cold-TNFα原核表达载体,完成了TNF-α重组蛋白的表达,建立了两步分离纯化方案,并利用Western Blot和细胞刺激实验检测了重组人TNF-α蛋白抗原性和生物学活性。[结果]利用大肠杆菌成功表达重组人TNF-α蛋白,经酶切纯化后的人TNF-α蛋白不含标签序列,纯度达95%,收率约70%,并具有良好的生物学活性。[结论]获得具有生物学活性的人TNF-α蛋白,为进一步的中和抗体研究奠定了基础。     

融合干扰素-BLA（IFN-BLA）的构建、表达、纯化及抗病毒活性测定

干扰素-BLA(IFN-BLA)由干扰素-beta-1b(IFN-beta-1b)和干扰素-alpha-2b（IFN-alpha-2b）通过连接肽-GGGS-融合而成。优化了其在大肠感菌BL21 CodonPlus (DE3)-RIL中的实验室表达条件,表达的目的蛋白占菌体总蛋白的35％以上并且主要以包涵体的形式存在。对包涵体的复性条件进行了摸索,建立了IFN-BLA的复性及纯化方法,纯化后的蛋白产量约为45 mg/L, 纯度在90％以上。抗病毒活性分析表明这一新的融合蛋白可能具有协同或加成活性。     

干扰素-tau的原核表达、纯化和活性测定

干扰素-tau (IFN-tau)是一种新发现的I型干扰素,为了更清楚的研究它的生物学功能,在已克隆IFN-tau cDNA的基础上,PCR扩增出IFN-tau ORF,与原核表达载体pBV220重组后,成功的构建了IFN-tau的原核表达质粒pBV220/IFN-tau。重组质粒转化大肠杆菌BL21,该菌经过诱导,用凝胶过滤色谱层析的方法获得了纯化的目的蛋白,该蛋白经过氨基酸序列分析证实是IFN-tau ;用细胞病变抑制法测定IFN-tau经过透析复性后的活性为2.09×106IU/ml,比活性为2.35×106IU/mg。     

基因工程犬干扰素α的制备及纯化工艺

目的：运用基因工程技术制备高活性的重组犬仪干扰素。方法：用发酵罐大量培养工程菌,经超声破碎菌体获粗制包涵体,用1％ TritonX-100洗涤去除部分杂蛋白后,以8mol／L尿素溶解包涵体,稀释复性并超滤浓缩,用阴离子柱层析法纯化目的蛋白,测定重组犬干扰素d的活性。结果：得到的重组犬干扰素仅的相对分子质量为19×10^3,蛋白含量为0.55mg／mL,纯度95.65％,目的蛋白回收率为13.75％,活性1.78×10^7U／mL,比活性3.24×10^7U／mg。结论：制备了高纯度且具有高活性的重组犬α干扰素。     

猪α-干扰素的原核表达及活性测定

目的：表达并纯化出具有生物学活性的重组猪α-干扰素。方法：根据前期合成的猪α-干扰素基因序列设计引物,用PCR方法扩增出猪α-干扰素基因,并将其定向克隆入原核表达载体pET28中,酶切及测序鉴定正确后,转化大肠杆菌BL21进行诱导表达,对表达产物通过镍琼脂糖凝胶柱亲和层析纯化、透析法复性后,采用微量细胞病变抑制法测定重组猪α-干扰素的活性。结果：测序结果表明构建了猪α-干扰素原核表达载体pET28-poIFN-α;诱导表达后经SDS-PAGE分析,在相对分子质量约21×10^3的位置出现明显的诱导蛋白条带,与目的蛋白大小相近;经分析表达产物主要以包涵体形式存在,约占菌体总蛋白的36%,纯化后的目的蛋白纯度约为90%;采用微量细胞病变抑制法测定其活性约为1.67×10^6U/mg。结论：获得了纯度较高并具有较高活性的重组猪α-干扰素,为下一步研究猪α-干扰素药物价值及其生物制剂的生产、应用奠定了基础。     

重组人干扰素α2b的提取和纯化

为了提高重组人干扰素α2b的质量 ,增加产量 ,降低成本 ,通过破菌 ,包涵体提取 ,离子交换层析 ,凝胶过滤等纯化方法制备干扰素纯品。结果表明干扰素纯度可达 95%以上 ,比活达到 1.2× 10 8IU/mg以上。结果提示 ,采用本方法收率高 ,纯度好 ,工艺稳定 ,适合大规模生产     

鸡γ干扰素的可溶性表达及纯化产物的抗病毒活性

【目的】克隆鸡γ-干扰素（chIFN-γ）基因,大肠杆菌表达及产物纯化与活性检测。【方法】通过RT-PCR方法用ConA刺激的20日龄SPF鸡的脾脏淋巴细胞中扩增出chIFN-γ成熟蛋白基因cDNA,克隆至原核表达载体pET-32a (+),构建重组表达质粒pET-32a (+)-chIFN-γ,在大肠杆菌BL21（DE3）中经IPTG诱导表达,利用镍柱亲和层析法纯化可溶性蛋白,并进行SDS-PAGE、Western blot鉴定。利用MDCK-VSV系统测定抗病毒活性。【结果】克隆得到456 bp 的chIFN-γ成熟蛋白编码基因,大肠杆菌中成功表达chIFN-γ蛋白,分子量约为31.0 kDa,能与抗His的单克隆抗体和兔源多抗血清发生特异性反应。表达蛋白一部分形成包涵体,另一部分以可溶形式存在,可溶性蛋白经镍柱在天然条件下的纯化得率为3.0 mg/mL。生物学活性试验表明,1:32 稀释纯化的重组chIFN-γ （rchIFN-γ）孵育MDCK细胞后能抵抗100TCID50的VSV攻击。【结论】通过镍柱天然纯化获得的rchIFN-γ具有较好的抗病毒活性,为研制新型抗病毒干扰素制剂奠定基础。     

Expression and purification of chicken beta interferon and its antivirus immunological activity

Interferon beta (IFN-β) belongs to a class of natural proteins that can inhibit virus replication. In this paper, nucleic acid sequence encoding chicken interferon beta (chIFN-β) mature protein was cloned and highly expressed in Escherichia coli (E. coli) by 1mM IPTG induction. The expressed chIFN-β was about 20% of total bacterial protein. The recombinant protein in form of inclusion body was then solubilised, refolded and purified to a purity of greater than 95% by immobilized metal ion affinity chromatography (IMAC). This recombinant chIFN-β could inhibit vesicular stomatitis Indiana virus (VSV) and infectious bursal disease virus (IBDV) replication in vitro. Animal experiments showed that the recombinant chIFN-β could decrease pathological lesions caused by the IBDV in bursa of Fabricius. These results will provide useful information for further investigations on veterinary clinical applications and fundamental research.     

Antiviral and antiproliferative activity of human fibroblast interferon fractions

The affinity chromatography of Human crude beta-interferon preparations on Blue Dextran Sepharose columns resulted in isolation of several fractions with different ratio of antiviral to antiproliferative activities. The results of investigation of two of these fractions are described in this report. The first of them was eluted by 1N NaCl in 0.01 M tris buffer at pH 7.8, the second was eluted by 1 M NaCl, 50% methylethylenglycol in 0.01 M tris-HCl buffer at pH 7.8. The first of the fractions possessed presumably antiproliferative and the second presumably antiviral activity. Both fractions induced the increase of 2'5'-oligoadenylatesynthetase activity in cells although the inducing activity of the first fraction was about 6-fold higher than that of the second one as compared with their antiviral activities. The obtained results indicate that purification of interferon preparation for interferons main antiviral activity may lead to the loss of the great part of antiproliferative material.     

Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5?mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1?l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195?Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC(50)?=?16-64?μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.     

hHO-1结构预测及突变体的构建、表达、纯化和活性检测

人血红素加氧酶-1(human heme oxygenase-1,hHO-1)是血红素代谢的限速酶,直接调节体内胆红素水平。利用软件Spdbv程序对hHO-1进行结构模拟预测,以Ala取代第25位His残基,hHO-1活性部位的结构有较明显的改变,但据模拟推测突变后hHO-1与血红素仍然具有结合性。依据模拟结果,构建野生型和突变体表达载体pBHO-1和pBHO-1(M),并分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白。表达产物经30％-60％(NH4)2SO4盐析后纯度提高3.6倍,再经两次Q-Sepharose Fast Flow阴离子交换树脂分离则表达产物的纯度提高30倍。酶活性测定显示,突变体hHO-1(△hHO-1)较野生型hHO-1(whHO-1)活性下降了91．21％。本研究显示hHO-1的第25位His在酶与底物血红素氧化反应中起着重要作用,为有效调控酶活性发挥其生物学作用提供依据。     

Design, synthesis and antiproliferative activity of novel aminosubstituted benzothiopyranoisoindoles

The synthesis of a number of new benzothiopyrano[4,3,2-cd]isoindole aminoderivatives designed as structural analogues of the key metabolite of the anticancer agent Ledacrine (nitracrine) and their in vitro cytotoxic activity evaluation against HCT-116, MES-SA, and MES-SA/Dx cancer cell lines is reported. The majority of the derivatives possessed noticeable cytotoxicity in a low μM range indicating an interesting structure-activity relationship.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

Synthesis and antiproliferative activity of some novel derivatives of diospyrin, a plant-derived naphthoquinonoid

Derivatisation of diospyrin, a bisnaphthoquinonoid isolated from Diospyros montana Roxb., led to the modification of its inhibitory activity, in vitro, towards a murine tumour model, Ehrlich ascites carcinoma (EAC), and two human cancer cell lines, viz., malignant skin melanoma (A375) and epidermoid laryngeal carcinoma (Hep2). Among the novel derivatives, an epoxide exhibited the maximum antiproliferative activity (IC(50) values in the range of 0.03-0.21 microM) and a comparatively lower toxicity (IC(50) approximately 98 microM) in normal human peripheral blood mononuclear cells (PBMC). This compound might provide a novel 'lead' for the development of clinically effective antiproliferative agents against cancer.     

Over expression and purification of recombinant human myocilin

Human myocilin is a 55 kDa protein that is implicated in primary open angle glaucoma (POAG). Understanding the structure and folding of the native protein and the mutants that increase aggregation could lead to possible prevention of the condition. We report here the over expression and purification of the human myocilin in E. coli. The initial expression of recombinant myocilin in E. coli was found to be low. The problem of low yield was found to be due to multiple causes and was overcome using a suitable combination of vectors, tags, host background and expression protocols. The overexpressed human myocilin was purified by affinity column chromatography to yield about 8 mg of protein from 1 l of culture. The protein purity and folding were confirmed using electrophoresis, immunoblotting and fluorescence spectroscopy. Further biophysical characterization and crystallization trials using the recombinant human myocilin will pave the way for better understanding of the structure–aggregation relationship that is involved in causing POAG.     

Design, synthesis, and in vitro antiproliferative activity of novel Dasatinib derivatives

Two series of novel Dasatinib derivatives have been designed and synthesized, with their in vitro cytostatic effect screened on human chronic myeloid leukemia cell line K562 and human myeloid leukemia cell line U937. Some target compounds demonstrated significant inhibitory activities against both cell lines. Compared to the contrast drug Dasatinib, 1b, 1c, 1d, 1e and 1f were found to demonstrate more potent antitumor activities. The structures of all the newly synthesized compounds were determined by (1)H NMR and (13)C NMR.