抗肿瘤疫苗MAGE1-MAGE3-HSP70蛋白特性及其纯化策略的研究

目的：对MAGE1-MAGE3-HSP70（M1-M3-HSP70）融合蛋白的理化性质及其纯化策略进行初步研究,为后期的临床前试验提供数据和依据。方法：以本实验室构建的菌种（BL21（DE3）pLysS-M1-M3-HSP70）为材料,采用常规细菌培养及诱导方法进行诱导表达,用聚丙烯酰胺凝胶（SDS—PAGE）电泳、免疫印迹（Westemblotting）对目的蛋白进行分析,然后确定盐析条件,使用ButylSepharoseHP疏水相互作用层析以及阴离子交换（Source30Q填料）对目的蛋白的纯化进行分析。结果：M1-M3-HSP70蛋白在大肠杆菌中表达后,目的蛋白分为两部分,一部分为包涵体形式（44．9％）,一部分为可溶表达（55．1％）。对其可溶部分进行研究发现．细菌裂解后,目的蛋白存在聚合和降解现象;确定了盐析条件、洗脱缓冲液以及稳定的PH值范围,基本确定了目的蛋白的纯4~．z-艺。结论：明确了M1-M3-HSP70融合蛋白的基本性质,确定了目的蛋白的纯化方法,为基于融合蛋白的肿瘤疫苗的进一步研究提供了参考。     

融合蛋白GST-Ulp1p在大肠杆菌中的高效可溶性表达及其活性鉴定

利用基因工程技术,体外重组小分子类泛素修饰蛋白酶1(Ulp1)的活性片段,获得高表达、高特异性重组蛋白酶。从酿酒酵母Saccharomyces cerevisia中提取Ulp1编码第403到621个氨基酸残基之间的DNA片段(Ulp1p),在其C端加入6×His并连接到大肠杆菌表达载体pGEX中,构建重组表达质粒pGEX-Ulp1p-his6。将重组质粒转化至大肠杆菌Rosetta(DE3)中,氨苄青霉素抗性筛选转化子。表达、纯化后,以SUMO融合蛋白检测其活性。经过优化,该蛋白可溶性表达,表达量占菌体总蛋白的40.12%。可通过谷胱甘肽琼脂糖凝胶柱或Ni-NTA凝胶亲和层析纯化得到纯度98%的蛋白。经酶切分析,比活力为1.375×104U/mg。融合蛋白GST-Ulp1p-His6无需切除谷胱甘肽S-转移酶(GST)标签,具有很高的活性,制备简易;6×His标签,有利于底物蛋白切割后纯化,减少蛋白损失。本研究为制备高活力的SUMO蛋白酶提供了一个新方法。     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

日本对虾c型溶菌酶的高效重组表达及产物分析

从日本对虾(Marsupenaeus japonicus)血液中提取总RNA,根据GenBank已登录的该cDNA序列(AB080238),通过RT-PCR技术扩增出日本对虾溶菌酶(MjLys)成熟肽基因。该基因完整的开放阅读框为477 bp,编码158个氨基酸(aa),前18 aa为信号肽,成熟肽由140 aa组成,分子量为16.4 kD,理论等电点(pI)为8.80。经分析表明,该基因含有一个完整的c型溶菌酶结构域(1-130 aa),包括c型溶菌酶特有的两个活性中心Glu33和Asp50,以及8个保守结构Cys残基。将MjLys成熟肽基因亚克隆至原核表达载体pET-32a(+),在大肠杆菌细胞BL21(DE3)pLysS中诱导发酵,实现了重组MjLys蛋白的高效表达,并测定了该重组蛋白对几种细菌的抑菌活性。结果表明,重组日本对虾溶菌酶对革兰氏阳性菌金黄色葡萄球菌和溶壁微球菌均有显著的溶菌活性。     

在大肠杆菌中表达有活性的人STK11蛋白

STK11蛋白(serine/threonine kinase11)是近年来发现的具有多种重要功能的蛋白,可参与调控细胞周期、p53介导的细胞凋亡、ras诱导的细胞转化、细胞极化等多种生物学过程。利用大肠杆菌高效表达有活性的人STK11蛋白,可为其结构和功能的深入研究打下良好基础。利用本室克隆的人STK11 cDNA和原核表达载体pET-44a( )构建带有Nus融合标签的诱导型表达载体pET-Nus-STK11,在不同的大肠杆菌宿主中诱导表达。SDS-PAGE和Western blot检测表明,在BL21(DE3)宿主中表达的融合蛋白主要以包涵体形式存在,占菌体总蛋白的8.9%;在Rosetta-gami(DE3)pLysS宿主中主要表达为可溶性蛋白,占菌体总蛋白的16.7%。而经纯化和包涵体蛋白复性处理后,以Chariot介导重组融合蛋白进入人肝癌细胞SMMC-7721检测其对细胞生长和细胞周期的影响。与对照组相比,BL21(DE3)中表达的Nus-STK11蛋白几乎无抑制活性;而Rosetta-gami(DE3)pLysS中表达的Nus-STK11蛋白可以显著抑制SMMC-7721细胞的生长,抑制率达47.05%,并导致细胞周期的G0/G1期阻滞,证实表达的重组融合蛋白具有明显的生物学活性。上述结果为在大肠杆菌中成功表达有活性的重组STK11蛋白的首次报道。     

小鼠RHOX5蛋白两种截短型突变体的原核表达及其与MDFIC互作的鉴定

目的：表达和纯化两种小鼠RHOX5蛋白的截短型突变体,确定完整的RHOX5蛋白及其两种截短型突变体与MDFIC蛋白结合的能力。方法：生物信息学分析小鼠同源异型框蛋白RHOX5的cDNA序列,分别对RHOX5的两种截短型片段RHOX5 N和RHOX5 C进行PCR、分别扩增RHOX5的两种截短型片段RHOX5 N和RHOX5 C并将其克隆至pGEX4T3原核表达载体,构建重组表达质粒。用重组表达质粒分别转化大肠杆菌RosettaTM2(DE3)菌株,经IPTG诱导后,使用Glutathione-Sepherase 4B颗粒对融合蛋白进行小批量亲和纯化,通过SDS-PAGE电泳分离目标蛋白,确定融合蛋白的表达。进行GST-pull down实验,检测完整的RHOX5蛋白及其两种截短型突变体与MDFIC蛋白结合的能力。 结果：在大肠杆菌RosettaTM2(DE3)中有效地实现了GST-RHOX5、GST-RHOX5 N和GST-RHOX5-C-3种融合蛋白的可溶性表达;经Glutathione Sepherase 4B颗粒亲和纯化后,获得了纯化后GST融合蛋白;GST-pull down实验证实,含有homeodomain的RHOX5蛋白和RHOX5C截短型突变体可以与MDFIC蛋白相结合,而RHOX5N截短型突变体则丧失了与MDFIC蛋白结合的能力。 结论：实现了RHOX5及其两种截短型突变体的原核表达和纯化,证实RHOX5蛋白的homeodomain结构域是其与MDFIC结合的关键部位。     

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.     

胆汁三烯结合蛋白在大肠杆菌中的表达、复性与纯化

目的：合成胆汁三烯结合蛋白（BBP）基因并在大肠杆菌中表达,获得重组BBP纯化制品。方法：根据天然BBP的基因序列和大肠杆菌偏好密码子设计并合成BBP基因的引物,PCR扩增优化的BBP基因序列,克隆至载体pEasy-T3;测序正确后,将该序列克隆至表达载体pET-32a上,构建表达质粒,转化至大肠杆菌BL21（DE3）pLysS,在IPTG诱导下表达融合蛋白;采用Ni柱纯化融合蛋白。结果：PCR扩增获得了优化后的BBP基因序列,构建了表达载体pET-32a-BBP;SDS-PAGE分析表明表达的融合蛋白相对分子质量为20×10^3,以包涵体形式存在,占全菌蛋白的40%以上;变性、复性后经Ni2＋柱纯化,获得纯度达98%以上的重组蛋白。结论：优化并合成了BBP全基因序列,获得了高纯度重组融合蛋白,为进一步鉴定其生物活性及筛选小分子的研究奠定了基础。     

IL-1023-57-PE40分泌表达的初步研究

将IL-1023-57-PE40基因与pelB信号肽融合置于pET-20b构建分泌表达质粒pET-20b-IL-1023-57-PE40,然后将pET-20b-IL-1023-57-PE40分别转化至BL21(DE3),BL21(DE3)pLysS,Rosetta(DE3),E·coliK12TB1,ER2566中。无论是在37℃或是在26℃,亦或在培养基中添加葡萄糖的情况下,IPTG诱导后,IL-1023-57-PE40蛋白只在BL21(DE3)pLysS菌中以可溶分泌形式表达,其中以37℃时培养基中不添加葡萄糖表达量为最高,占菌体蛋白总量的15%,说明蛋白的分泌表达与菌种的选择有关。表达产物经免疫印记检测可被抗PE40的特异抗体识别。通过质粒稳定性实验证明,pET-20b-IL-1023-57-PE40在BL21(DE3)中不稳定,导致蛋白的不表达,在Rosetta(DE3)BL21,E·coliK12TB1,ER2566中稳定但不表达,因此,以Rosetta(DE3)BL21为例,通过SDS-PAGE、DNAStar和ANThewin蛋白分析软件对本室构建的几种PE重组毒素进行比较分析,我们发现:并不是所有PE重组毒素融合信号肽序列后,就能分泌表达,PE重组毒素分泌表达还可能与导向部分的性质有关。     

玉米黑粉菌cyp51基因结构分析与克隆表达

通过生物信息学手段分析cyp51基因结构,并根据GenBank登记的玉米黑粉茼cyp51 DNA序列,设计cyp51引物和两对分别截短不同跨膜区的突变体引物,构建了多种重组表达质粒及突变体重组表达质粒.选用不同宿主菌包括Escherichia coli BL21(DE3).BL21(DE3)pLysS和Rosetta(DE3)诱导表达并优化条件.SDS-PAGE分析结果表明:只有突变体pET32-YH-35能够在E. coli BL21(DE3)中高效表达(30℃,0.5 mmol/L IPRG诱导).通过与戊唑醇等4种商品化杀菌剂农药和14种XF系列农药先导化合物的紫外结合光谱分析表明:重组蛋白具有生物学活性.其中一种XF系列化合物的结合常数接近商品化杀菌剂,有可能开发为新的杀菌剂,为设计开发新型高效抗真菌新药提供了理论依据.     

人巨细胞病毒结构蛋白p150、p38的原核表达纯化及免疫性鉴定

应用聚合酶链式反应(PCR)扩增HCMV结构蛋白p150aa595~aa614/aa1006~aa1048和p38aa117~aa373基因片段,将其分别克隆于表达载体pGEX-4T-1和pET30 a-SetB中,转化大肠杆菌BL21(DE3),IPTG诱导表达,重组质粒经酶切鉴定及DNA测序证实pGEX-4T-1/p150aa595~aa614/aa1006~aa1048和pET30 a-SetB/p38aa117~aa373构建正确;通过优化表达和纯化,分别获得分子量约为35kD和43kD纯化的表达产物。经亲和层析纯化以免疫印迹实验对纯化的重组蛋白进行免疫反应性检测,证实重组蛋白p150aa595~aa614/aa1006~aa1048、p38aa117~aa373能够与抗HCMV IgM阳性血清反应,说明表达的重组蛋白p150aa595~aa614/aa1006~aa1048、p38aa117~aa373具有良好的免疫性,对HCMV的原发感染具有潜在的应用价值。     

Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

表达大肠杆菌K88ac-ST1-LTB融合蛋白基因工程菌株的构建

利用PCR技术,从大肠杆菌C83902质粒中扩增出K88ac基因、ST1突变基因和LTB基因,通过分离、纯化、内切酶酶切、连接和转化,构建了含K88ac-ST1-LTB融合基因表达载体的重组菌株BL21（DE3）（pXKST3LT5)。经酶切鉴定和DNA序列分析证实,构建的重组质粒pXKST3LT5中含有K88ac-ST1-LTB融合基因,且基因序列和阅读框架均正确。经ELISA检测,重组菌株表达的K88ac-ST1-LTB融合蛋白能够被ST1单抗、LTB和K88ac抗体识别。经乳鼠灌胃试验证实,表达的融合蛋白已丧失天然ST1肠毒素的活性。免疫实验结果表明,K88ac-ST1-LTB融合蛋白能够诱发小白鼠产生抗体,该抗体具有中和天然ST1肠毒素的毒性作用,表明构建的重组菌株可以作为预防仔猪黄、白痢基因工程菌苗的候选菌株。     

High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni²⁺ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.     

基于谷氨酸棒杆菌NCgl1221蛋白的新型细菌表面展示系统

【目的】开发一种新型的大肠杆菌表面展示系统,为C末端截短NCgl1221蛋白作为锚定蛋白提供科学依据,丰富并优化细菌表面展示系统。【方法】扩增C末端截短NCgl1221序列和β-淀粉酶基因,构建融合蛋白表达载体。将重组载体PET-NA和空载体PET-28a分别转入Rosetta(DE3)pLysS中,IPTG诱导表达,SDS-PAGE和Western blot鉴定融合蛋白表达情况。将诱导表达菌株进行免疫荧光染色,荧光显微镜观察和流式细胞分析检测β-淀粉酶的展示。酶活测定和淀粉水解分析验证被展示β-淀粉酶的活性。【结果】融合蛋白成功地在大肠杆菌中表达,有活性的β-淀粉酶通过与锚定蛋白C末端的融合被展示在了宿主菌表面,展示β-淀粉酶的重组菌可以水解利用培养基中的淀粉。【结论】成功开发了一种以C末端截短NCgl1221为锚定蛋白的新型大肠杆菌表面展示系统,并以此系统展示了分子量大小为56 kDa的活性酶,为该系统在全细胞催化剂或吸附剂等方面的应用奠定了基础。     

人工锌指核酸酶的设计与表达纯化

设计表达了四个锌指核酸酶,用于切断人基因组中的rRNA基因家族的内部转录间隔序列,造成双链断裂,以此提高针对多位点基因打靶的效率,为后续基因打靶应用于基因治疗研究奠定基础。首先,在人rRNA基因家族ITS1序列中找到两个合适的9 bp长的序列(中间间隔6 bp)为锌指蛋白识别位点,根据识别位点序列每个位点分别设计两个三锌指蛋白。通过设计引物进行重叠延伸PCR得到全长编码锌指蛋白的DNA,分别克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFP,转化大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指融合蛋白的表达与纯化。同时,将限制性内切酶Fok I的切割结构域分别与四个锌指蛋白序列采用PCR拼接后克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFN,转化到大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指核酸酶融合蛋白的表达并纯化。     

带有穿膜结构域的转录因子蛋白Oct4和Sox2的融合表达

目的:克隆、表达及纯化带有穿膜结构域的转录因子蛋白Oct4和Sox2。方法:根据GenBank中的Oct4和Sox2基因序列,在其3’端引入穿膜结构域11R,并在其两端引入NdeⅠ和XhoⅠ酶切位点,进行全基因合成;将目的基因克隆至pET41a载体,进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21(DE3),经IPTG诱导表达后,对表达产物进行Western印迹鉴定;最后用Ni-NTA亲和层析柱对所获目的蛋白进行纯化。结果:质粒酶切鉴定结果表明带有目的基因的重组质粒构建成功;SDS-PAGE结果显示有相对分子质量约42×103和38×103的特异性蛋白表达条带,经Western印迹证实为目的蛋白;用Ni-NTA亲和层析柱纯化后,得到均一的Oct4和Sox2目的蛋白。结论:得到带有穿膜结构域的转录因子融合蛋白Oct4和Sox2,为今后安全开展诱导性多能干细胞研究奠定了基础。     

人血管生成素-PE40融合蛋白基因的构建、表达及活性研究

利用 PCR扩增出人血管生成素 (h ANG)成熟肽的基因片段 .与绿脓杆菌外毒素缺失突变体PE40的基因连接后 ,克隆入 p UC1 9载体中 .测序后克隆入表达载体 p RSETB,构建成 h ANG-PE40融合基因的表达载体 .IPTG诱导 ,表达出分子量约为 58k D的 His6- ANG- PE40融合蛋白 ,占菌体总蛋白的 8% .Ni2 +- NTA树脂纯化表达蛋白 ,SDS- PAGE结果显示纯化重组蛋白为单一条带 .鸡胚绒毛尿囊膜鉴定表明重组蛋白体外能够有效地抑制血管的形成 .