葡萄球菌肠毒素 B 突变体的构建及其抗肿瘤活性分析

目的：通过对葡萄球菌肠毒素B（SEB）32位His进行定点突变,获得抑瘤效果显著增强的SEB突变体。方法：利用基因定点突变的方法,将SEB的32位His替换为Asn,将重组质粒转入大肠杆菌中诱导表达,用CM弱阳离子层析柱纯化获得重组蛋白,用SDS-PAGE和Western印迹对其进行鉴定,并用增殖实验检测其丝裂原活性,通过MTS法检测其体外抗肿瘤活性。结果：构建并高效表达了突变体蛋白SEB（H32N）,纯化获得了足够纯度的突变体蛋白;体外实验表明,在相同浓度下,SEB（H32N）的丝裂原活性及体外抗肿瘤活性明显优于野生型SEB。结论：同野生型SEB相比,突变体蛋白SEB（H32N）对肿瘤生长的抑制作用得到了提高。     

增强型金黄色葡萄球菌肠毒素C2突变体及其超抗原活性

金黄色葡萄球菌肠毒素C2 (Staphylococcal enterotoxin C2,SEC2)作为一种超级抗原蛋白,极微量即可有效激活机体免疫系统,这一特性可应用于对肿瘤和感染性疾病的辅助治疗.为了增强SEC2的超抗原活性,应用over-lap PCR方法将SEC2中的102～106位GKVTG氨基酸残基分别突变为WWH、WWT和WWP,获得3种突变体ST-1、ST-2和ST-3.三种突变体刺激小鼠淋巴细胞增殖活性和肿瘤细胞生长抑制活性与野生型SEC2相比均有显著提高.ST-1和ST-3的致热活性与野生型SEC2相当,而ST-2的致热活性明显高于野生型SEC2.此外,三种突变体体外刺激淋巴细胞分泌IL-2、IFN-γ和TNF-α的水平也显著提高,这可能是导致突变体具有较高肿瘤细胞生长抑制活性的原因.进一步实验发现,三种突变体刺激下,小鼠脾淋巴细胞mVβ8.2基因的转录水平较野生型SEC2显著增加,暗示突变体对TCR mVβ8.2分子亲和力的提高,可能是其超抗原活性增强的主要原因.     

肠毒素C2蛋白C,N末端对其活性的影响

目的:分析金黄色葡萄球菌肠毒素C2(SEC2)中N,C末端对其超抗原活性和可溶性表达能力的影响。方法:应用基因工程技术对SEC2的N,C末端进行部分删除,获得三种突变蛋白,并对其进行体外超抗原活性和可溶性表达能力的比较。结果:对SEC2的N,C末端的删除都在一定程度上影响其超抗原活性和可溶性表达能力,其中,N末端的两个删除突变体的超抗原活性分别降低40%和48%,而删除C末端则使其可溶性表达水平下降到野生型的20%左右。结论:SEC2蛋白分子的N末端对其超抗原活性起主要作用,C末端对其可溶性表达具有显著影响,而完整的SEC2分子对于其发挥最大生物学活性是必要的。     

肠毒素C2中Met24对其超抗原活性无重要作用

目的:分析金黄色葡萄球菌肠毒素C2的T细胞受体结合区域α3结构中,第24位甲硫氨酸对其超抗原活性的影响作用.方法:应用定点突变技术对金黄色葡萄球菌肠毒素C2的Met24进行无意义突变,获得突变蛋白SEC2(M24A),对突变蛋白和野生型蛋白的体内、体外超抗原活性进行比较.结果:分别对野生型蛋白及突变蛋白的免疫刺激活性、肿瘤抑制活性、体内致热毒性进行了比较,发现突变蛋白的增值指数(PI)、抑瘤率、热源效应与野生型相比无显著差异(P＞0.05),这表明对第24位甲硫氨酸的替换没有对金黄色葡萄球菌肠毒素C2的超抗原活性造成明显影响.结论:在金黄色葡萄球菌肠毒素C2的α3结构中,Met24并不是决定其超抗原活性的重要氨基酸残基.     

FABP5基因重组突变体蛋白抗前列腺癌细胞活性分析

为构建脂肪酸结合蛋白5 (FABP5)突变体的原核表达体系,评价突变体蛋白质体外抗前列腺癌细胞的活性.利用定点突变技术,突变FABP5蛋白脂肪酸结合的3个关键位点,并构建原核表达体系,对重组蛋白质进行原核表达、分离纯化.通过细胞毒性、细胞划痕和细胞侵袭试验,评价重组FABP5突变体蛋白质对前列腺癌细胞22RV1和PC3增殖、迁移和侵袭的影响.结果 显示定点突变后的DNA与表达载体pQE32连接并转入大肠杆菌(Escherichia coli) BL21(DE3),经序列测定正确,构建了重组表达工程菌,并诱导表达的重组蛋白经亲和层析纯化获得纯度较高的重组蛋白;三突变体对细胞的增殖、迁移和侵袭抑制作用比3个单突变体和3个双突变体抑制效果明显;单突变体和双突变体组内对细胞的增殖、迁移和侵袭抑制作用差异较小;而野生型重组蛋白质对两株细胞的增殖、迁移和侵袭具有促进作用.本研究从所有突变体中筛选出FABP5的三突变体重组蛋白质对前列腺癌细胞抑制作用较好,为后续开发去势抵抗性前列腺癌(CRPC)蛋白质药物提供参考.     

P48L和D74N突变对p16基因功能的影响

应用DNA聚合酶链式反应(PCR)技术,对p16抑癌基因(CDKN2)进行体外定点突变,在p16cDNA中引入第48位密码子CCG(Pro)→CTG(Leu)和第74位密码子GAC(Asp)→AAC(Asn)突变,构建了p16-P48L和p16-D74N突变体。野生型和突变型p16 cDNA克隆于pcDNA3构建pCMV-p16、pCMV-p16P48L和pCMV-p16D74N真核表达载体,导入纯合缺失p16基因的人肺癌细胞株H460,经RNA点杂交、RNA印迹和细胞免疫化学染色,检测到P16表达。通过比较表达野生型和突变型P16的H460细胞在~3H-TdR掺入及细胞所在周期的差异,证实P16表达抑制细胞进入S期,而P48L和D74N突变体对细胞进入S期没有什么影响,提示P48L和D74N突变导致P16蛋白功能丧失。     

活性增强的SEC2截短突变蛋白的构建及其生物活性研究

目的:分析金黄色葡萄球菌肠毒素C2的截短蛋白SEC14-239中7和9位氨基酸对其超抗原和抗肿瘤活性的影响.方法:利用基因工程方法将SEC14-239中7和9位Thr和Gly分别替换为Leu和Glu后,获得截短突变蛋白SEC14-239M.体外细胞学实验对其超抗原活性和抗肿瘤活性进行分析.结果:突变蛋白SEC14-239M体外刺激小鼠T细胞增值活性较SEC14-239显著增强(P＜0.05),与未截短的SEC2相当(P＞0.05).在相同剂量条件下,SEC14-239M诱导的抗肿瘤活性尽管低于SEC2(P＜0.01),但要显著高于SEC14-239(P ＜0.05).结论:截短蛋白SEC14-239的第7和9位氨基酸突变后能显著增强其超抗原活性和抗肿瘤活性,但二者增强程度有差异.这一结果说明以定点突变改造SEC2截短体的活性是可行的,同时也暗示超抗原的T细胞增殖和诱导肿瘤细胞抑制并不完全关联.     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

金黄色葡萄球菌肠毒素 C2 的基因克隆、 表达及其生物学活性

利用 PCR 技术从金黄色葡萄球菌的基因组 DNA 中克隆 SEC2 全长基因, PCR 产物与 pGEM-T 载体连接,经测序证实后进行亚克隆,构建其表达载体 pET-28a-SEC2 ,在大肠杆菌 BL21(DE3) 中表达成熟重组蛋白 (rSEC2) , 纯化 rSEC2 蛋白并对其生物学活性进行研究 . 结果表明：成功克隆了 SEC2 全长基因,测序证实该基因共 717 bp ,编码 239 个氨基酸,与 GenBank 中收录的 SEC2 成熟蛋白质序列完全一致, SEC2 基因登录 GenBank(Accession number ： AY450554) ; 构建了 SEC2 的表达载体 pET-28a-SEC2 ,并在大肠杆菌 BL21(DE3) 中得到高效可溶性表达,可溶性的 rSEC2 经 Ni²⁺ 亲和层析纯化达到电泳纯,纯化的 rSEC2 蛋白经蛋白质印迹检测,并能有效刺激人外周血单个核细胞的增殖,被 rSEC2 刺激的外周血单个核细胞在体外对肿瘤细胞的生长有显著的抑制作用 .     

低毒且高效杀伤人肿瘤细胞的新型人肿瘤

采用定点突变的方法构建了两株新型人肿瘤坏死因子突变体MT1 (32Trp157Phe)和MT2(2Lys30Ser32Trp157Phe), 并在大肠杆菌中得以高效表达, 表达量占菌体总蛋白的50%且为可溶性蛋白. 表达产物经蛋白印渍法检测, 均能与抗野生型hTNF-a单抗结合且经分离纯化后样品的纯度在95%以上. 各突变体虽然对鼠成纤维瘤细胞L929的生物学活性比野生型下降了4~5个数量级, 但却能高效杀伤人肿瘤细胞, 杀伤程度与野生型相差不明显. 测定突变体MT1的LD50值降至野生型的0.34%, 而MT2对小鼠30%致死剂量则低于野生型LD50的1/700.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> enterotoxin C2 mutants: biological activity assay in vitro

Staphylococcal enterotoxin C2 (SEC2) is one member of bacterial superantigens produced by Staphylococcus aureus. It can be attributed to its superantigenic activity to cross-link major histocompatibility complex class II molecules with T-cell receptors and activate a large number of resting T cells resulting in release of massive cytokines, which will produce significant tumor inhibition in vivo and in vitro. However, it could be not broadly applied to cure malignant tumors in clinic because of emetic activity of SEC2. The aim of this study was to inactivate emetic activity of SEC2 through site-directed mutagenesis. Cys93, Cys110 and His118 were selected as substitutional sites based on the functional sites responsible for emesis. The mutated proteins were used to determine Peripheral blood mononuclear cell proliferation activity and anti-tumor activity in vitro. Results showed that these mutated proteins efficiently stimulated T cell and exhibited the same tumor-inhibition effect as SEC2. It is possible to inactivate emetic activity of SEC2 through site-directed mutagenesis and provide satisfying agents for tumor treatment in clinic.     

新型截短肠毒素C2突变株抑制肿瘤细胞生长

在恶性肿瘤的治疗中,肠毒素C2 (SEC2) 的临床应用由于其副作用而被严重限制。利用SEC2基因截短技术,获得保留T细胞刺激活力又不引发催吐效应的SEC2突变株,可有效解决这个问题。根据噻唑蓝比色法 (MTT) 分析结果,新型截短肠毒素C2突变株 (NSM) 可显著刺激T细胞增殖,并且可显著抑制人大肠癌细胞 (CX-1) 和人乳腺癌细胞 (MCF-7) 生长。NSM的T细胞刺激能力和抑瘤效果与SEC2相似。动物研究结果证明NSM不再引发呕吐效应,且可显著抑制荷瘤小鼠的肿瘤生长。因此,这种可抑制肿瘤细胞生     

Enhancement of superantigen activity and antitumor response of staphylococcal enterotoxin C2 by site-directed mutagenesis

Bacterial superantigen staphylococcal enterotoxins (SEs) tremendously stimulate polyclonal T cells bearing particular TCR Vβ domains when binding to MHC II molecules, suggesting that they could be a candidate of new antitumor agent. SEC2, an important member of superantigen family, has been used in clinical trial as an immuntherapy agent for cancer treatment in China, and obtained some encouraging effects. However, the presence of immunosuppression and endotoxic activity limits the therapeutic dosage of SEC2, and influences its antitumor effect in clinic. Therefore, the enhancement of superantigen activity and antitumor effect of SEC2 could effectively make compensation for the disadvantages mentioned above. In this study, a superantigen SEC2(T20L/G22E) mutant was generated by site-directed mutagenesis, and efficiently expressed in E. coli BL21(DE3). The results showed that SEC2(T20L/G22E) mutant exhibited a significantly enhanced superantigen activity and antitumor response, compared with native SEC2 in vitro. Further toxicity assay in vivo indicated that SEC2(T20L/G22E) mutant had no significant increase in emetic and pyrogenic activity compared with SEC2, which suggested that the mutant SEC2(T20L/G22E) could be used as a potentially powerful candidate for cancer immunotherapy, and could make compensation for the deficiency of native SEC2 in clinic.     

一种白细胞介素2基因变构体的高效表达与纯化工艺

利用定点诱变技术构建表达质粒pET15b-MhIL-2并将其在大肠杆菌中进行表达发酵的优化研究,高效表达出可溶性的MhIL-2重组蛋白。蛋白经过亲和层析、Thrombin酶切、离子交换层析和凝胶过滤层析纯化,MhIL-2纯度达95%,且MhIL-2比hIL-2具有更强的促进T细胞增殖生物活性。     

Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis

All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.     

大肠杆菌3-酮基脂酰ACP还原酶110位天冬酰胺突变后的结构与功能

3-酮基脂酰ACP还原酶催化3-酮基脂酰ACP还原为3-羟基脂酰ACP,是细菌脂肪酸合成反应的关键酶之一.为了明确该酶中110位的保守天冬酰胺残基在酶催化活性和酶结构中的作用,本研究采用基因定点突变和蛋白质表达纯化技术,获得了大肠杆菌3-酮基脂酰ACP还原酶FabG的两个突变蛋白：FabG N110Q和FabG N110L.圆二色谱结果显示,天冬酰胺残基的突变改变了FabG的空间结构,使突变蛋白的α螺旋结构明显增加.以3-酮脂酰ACP为底物的酶活性测定表明,突变蛋白的酶活性均有下降,但残存的酶活性达到了FabG的75%以上.突变蛋白FabG N110Q和FabG N110L具有3-酮基脂酰ACP还原酶的活性,能在体外重建细菌脂肪酸合成反应.对fabG温度敏感突变株的遗传互补分析表明,FabG蛋白110位天冬酰胺突变为谷氨酰胺或亮氨酸后,在一定的条件下仍能互补大肠杆菌的生长.本研究结果提示,FabG 110位的天冬酰胺残基不是参与3-酮基脂酰ACP还原酶催化反应的必需氨基酸,它只是作为结构氨基酸,在维持FabG的空间结构的稳定性方面起作用.     

人干扰素α1b突变体IFNα1b/31K的构建及其生物学评价

目的:构建干扰素α1b突变体IFNα1b/31K,以期获得高效低毒的新型药物分子。方法:根据合理药物设计,采用定点突变技术,将干扰素α1b第31位氨基酸残基突变为K,并构建表达IFNα1b/31K重组蛋白。纯化后,对其抗病毒活性、抗肿瘤细胞增殖活性和动物体内急性毒性进行考察。结果:IFNα1b/31K表达量占菌体总蛋白的30%以上。纯化后的IFNα1b/31K纯度大于95%,比活性约为IFNα1b的1.7倍,抗肿瘤增殖活性比IFNα1b降低,未见对实验动物的急性毒性作用。结论:成功设计构建并表达了高效低毒的IFNα1b突变蛋白分子。     

Potent anti-tumor effects of an active site mutant of human manganese-superoxide dismutase. Evolutionary conservation of product inhibition

Mn-SOD serves as the primary cellular defense against oxidative damage by converting superoxide radicals (O(2)(-)) to O(2) and H(2)O(2). A unique characteristic of this mitochondrial anti-oxidant enzyme is the conservation from bacteria to man of a rapidly formed product inhibited state. Using site-directed mutagenesis, we have generated an active site mutant (H30N) of human Mn-SOD, which exhibits significantly reduced product inhibition and increased enzymatic efficiency. Overexpression of the H30N enzyme causes anti-proliferative effects in vitro and anti-tumor effects in vivo. Our results provide a teleological basis for the phylogenetically invariant nature of position His-30 and the evolutionary conservation of product inhibition. These data also provide more direct intracellular evidence for the signaling role associated with H(2)O(2).     

An engineered superantigen SEC2 exhibits promising antitumor activity and low toxicity

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic.