Zonula occludin toxin,a microtubule binding protein

AIM To investigate the interaction of Zot withmicrotubule.METHODS Zot affinity column was applied topurify Zot-binding protein（s）from crudeintestinal cell lysates.After incubation at roomtemperature,the column was washed and theproteins bound to the Zot affinity column wereeluted by step gradient with NaCl(0.3 mol·L-1-0.5mol·L-1).The fractions were subjected to6.0%-15.0%（w/v）gradient SDS-PAGE andthen transferred to PVDF membrane for N-terminal sequencing.Purified Zot and tauprotein were blotted by using anti-Zot or anti-tauantibodies.Finally,purified Zot was tested in anin vitro tubulin binding assay.RESULTS Fractions from Zot affinity columnyielded two protein bands with a Mr of 60 kU and45kU respectively.The N-terminal sequence ofthe 60 kU band resulted identical to β-tubulin.Zot also cross-reacts with anti-tau antibodies.Inthe in vitro tubulin binding assay,Zot co-precipitate with Mt,further suggesting that Zotpossesses tubulin-binding properties.CONCLUSION Taken together,these resultssuggest that Zot regulates the permeability ofintestinal tight junctions by binding tointracellular Mt,with the subsequent activationof the intracellular signaling leading to thepermeabilization of intercellular tight junctions.

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

Study on Spatial-temporal Dynamics of Bt Toxic Protein Expression in Insect-resistant Transgenic Cotton and Its Degradation in Soil

[Objective] This study aimed to investigate the spatial-temporal dynamics of Bt toxic protein expression in insect-resistant transgenic cotton and its degradation in soil. [Method] Btcry1Ac toxic protein expression in roots, stems and leaves of transgenic cotton Guoshen GK45 at different developmental stages and the annual average content of BtCry1Ac toxin protein in the topsoil, rhizosphere soil and following cotton-growing area were explored and analyzed by using enzyme linked immuno sorbed assay (ELISA). [Result] The content of exogenous BtCry1Ac toxin protein decreased during the growth process of insect-resistant transgenic cotton; to be specific, the content of BtCry1Ac toxin protein in cotton stems and leaves decreased more slowly and always maintained a high level, while that in roots decreased rapidly and reached a minimum level to the following plant growth and development stage. BtCry1Ac toxin protein was detected in topsoil of both non-transgenic and transgenic cotton-growing areas, and the content of BtCry1Ac toxin protein increased in topsoil of following cotton-growing area, which was very low in rhizosphere soil. [Conclusion] Determination of Btcry1Ac toxic protein provides scientific basis for the risk assessment of the cultivation of genetically modified crops and the safety evaluation of soil ecosystem.

Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis

Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.

Reporter Gene Assay for Detection of Shellfish Toxins

Objective To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry. Methods A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfcctants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software. Results Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL. Conclusion pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

Functional verification of the diphtheria toxin A gene in a recombinant system

Diphtheria toxin A （DTA）, a segment of the diphtheria toxin （tox）, inhibits protein synthesis in cells. When released from a cell, DTA is nontoxic and cannot enter other cells independently without the help of diphtheria toxin B. In this study, we artificially synthesized the DTA gene sequence and cloned it into pEGFP-N1 to generate the recombinant vector pEGFP-N1-DTA. This recombinant vector was then transfected into 293T cells to observe the effect of DTA protein expression on enhanced green fluorescent protein （EGFP） protein expression and the proliferation of 293T cells. After 48 h, high levels of EGFP expression were seen in control pEGFP-Nl-transfected cells, whereas very low levels were seen in cells transfected with pEGFP-N1-DTA. Reverse transcription polymerase chain reaction confirmed the expression of the DTA gene in cells transfected with pEGFP-N^-DTA. Further, the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （M-I-I-） assay revealed a significant difference in cell proliferation between the control group and the pEGFP-N1-DTA-transfected group. Using the expression of EGFP expression as an indicator, this study revealed that DTA expression can inhibit intracellular protein synthesis and cell proliferation.

基于Bt毒素的杀虫蛋白理性设计与创新应用策略

Bt毒素是源于苏云金芽孢杆菌(Bacillusthuringiensis)的具有特殊杀虫功能的大分子蛋白,其制剂和转基因作物已广泛用于害虫防治,产生了巨大的经济和社会生态效益。围绕Bt毒素挖掘和提升其应用价值是持续研究的热点,特别是随着Bt毒素结构功能和作用机制日趋明晰,为其功能修饰和创新应用创造了条件,相关研究蓬勃发展,成效显著。大量研究表明,采用定点突变、结构域替换或融合以及抗独特型抗体模拟等策略,是理性设计活性更高、稳定性更强、杀虫谱更广、非靶标生物安全性更高甚至是可用于害虫抗药性治理的有别于母体Bt毒素的突变体、结构杂合体乃至功能效应物抗体等新型杀虫蛋白的有效手段;此外,采用催化毒素活化、驱动毒素靶向受体结合、促进毒素表达以及同源或异源杀虫材料复配或共表达的协同促效等创新增效策略,也是助推Bt毒素应用价值的重要手段。本文总结了Bt毒素结构功能和作用机制,梳理了基于Bt毒素功能修饰的突变体、结构杂合体以及功能效应物抗体等新型杀虫蛋白理性设计和基于Bt毒素功能增效的创新应用策略等相关研究进展,并结合作者团队在模拟Bt毒素杀虫功能效应物抗体靶向设计研发方面的最新成果,探讨了基于Bt毒素的杀虫蛋白理性设计与创新应用策略未来发展动向及潜在可行捷径,为相关研究提供较为全面的最新有价值的文献资料和启发思路。

尿液中蛋白结合毒素水平与上尿路结石的相关性研究

目的·探讨尿液中蛋白结合毒素(protein-bound uremic toxin,PBUT)水平与上尿路结石形成的关系。方法·以海南省儋州市海头镇社区18~80岁居民为研究对象。记录研究对象基本信息和近3 d饮食情况,收集空腹血清和24 h尿液标本,所有对象接受肾脏和输尿管B超检查。将B超检查发现有上尿路结石或有明确上尿路结石病史的对象作为结石组,其他作为非结石组。检测血、尿中结石形成相关的生化指标,通过高效液相色谱检测血、尿中的PBUT(硫酸吲哚酚、3-吲哚乙酸和硫酸对甲酚),以及尿草酸和尿枸橼酸水平。通过多因素Logistic回归分析上尿路结石形成的相关因素。采用Spearman相关性检验分析尿PBUT与尿尿酸、草酸和枸橼酸的相关性。结果·经筛选,共纳入117例研究对象,其中结石组54例,非结石组63例。2组的性别、年龄、血清指标,以及高血压、糖尿病、高尿酸血症/痛风等合并症比例方面差异均无统计学意义。结石组的24 h尿液pH值、钙、尿酸和氯均显著高于非结石组(均P<0.05),而硫酸吲哚酚显著低于非结石组(P<0.05)。多因素Logistic回归分析结果显示,除尿钙外,尿硫酸吲哚酚水平(OR=0.929,95%CI 0.875~0.986,P=0.016)也是上尿路结石形成的独立相关因素。Spearman相关性分析结果显示,24 h尿液中3-吲哚乙酸(r=0.420,P=0.000)和硫酸对甲酚(r=0.307,P=0.001)水平均与草酸呈正相关,硫酸对甲酚水平与尿酸呈正相关(r=0.297,P=0.002),硫酸吲哚酚水平与枸橼酸呈正相关(r=0.289,P=0.002)。结论·在该研究人群中,尿硫酸吲哚酚水平降低可能是上尿路结石形成的独立危险因素,且尿液中PBUT水平与尿酸、草酸、枸橼酸的水平相关。

昆虫对Bt作物的抗性机制以及治理策略的研究进展

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)是一种分布十分广泛的革兰氏阳性菌,其在营养生长阶段和产孢阶段能够产生一系列杀虫蛋白,是农业、林业以及公共卫生实践中常用的生物防治剂。基于基因工程技术研制的Bt作物为农业害虫治理提供了一种高效、环保的生物防控手段。然而,长时间广泛种植Bt作物使得害虫逐渐产生Bt抗性,这大大降低了Bt蛋白的杀虫效果和Bt作物的长期效益。为了深入了解Bt蛋白的作用机制和昆虫Bt抗性产生之间的关系,本文归纳了多种Bt毒素的结构与功能特征,并从免疫系统调节、毒素激活变化和毒素受体基因突变三个主要方面阐述了昆虫Bt抗性发生的分子机制。此外,还介绍了基因堆叠和“高剂量/庇护所”这两种昆虫抗性治理策略。最后,指出Bt作物未来的研究方向,包括进一步深入解析Bt毒素的作用模式和昆虫Bt抗性发生的机制,发掘新的Bt蛋白,加强Bt作物科普宣传以及建立抗性监测网络和预警系统。

枳实消痞颗粒对脾虚气滞型腹膜透析患者营养不良及蛋白结合毒素的影响

目的观察枳实消痞颗粒对脾虚气滞型腹膜透析患者营养不良及蛋白结合毒素的影响。方法选取2021年10月—2022年10月在陕西中医药大学附属医院肾病二科及门诊随访的维持性腹膜透析营养不良患者60例,将其随机对照原则分为对照组和治疗组各30例。对照组采用西医基础治疗,治疗组在对照组治疗基础上服用枳实消痞颗粒,治疗3个月后观察肾功能指标及透析充分性指标[血肌酐(SCr)、尿素氮(BUN)、尿素清除指数(Kt)]、营养状态指标[血红蛋白(Hb)、血清白蛋白(ALB)、血清前白蛋白(PA)、体质指数(BMI)、肱三头肌皮褶厚度(TSF)、上臂围(AC)、上臂肌围(AMC)]、蛋白结合毒素指标[血清和腹透液中硫酸吲哚酚(IS)、硫酸对甲酚(pCS)水平]、中医证候积分、改良主观综合性营养评估表(MQSGA)评分的变化。结果治疗3个月后,治疗组SCr水平、BUN水平、血清和腹透液中IS水平、血清pCS水平及中医证候积分、MQSGA评分均较治疗前明显降低(P均<0.05),Kt及Hb、ALB、PA水平均较治疗前明显提高(P均<0.05),其余指标变化不明显(P均>0.05);对照组仅SCr水平明显降低(P<0.05);治疗后治疗组ALB、PA水平均明显高于对照组(P均<0.05),血清pCS水平及中医证候积分均明显低于对照组(P均<0.05)。结论枳实消痞颗粒能够改善脾虚气滞型腹膜透析患者的营养状态,降低血清及腹透液中IS水平和血清中pCS水平,具有保护肾脏和腹膜作用。

不同血液灌流治疗频率对维持性血液透析患者蛋白质能量消耗的影响

目的 探究不同血液灌流(hemoperfusion,HP)频率对维持性血液透析(maintenance hemodialysis,MHD)患者蛋白质能量消耗(protein-energy wasting,PEW)的影响。方法共纳入80例患者,根据HP治疗频率分为无HP(A)组、每月1次HP组(B)组、每半月1次HP(C)组、每周1次HP(D)组,每组20例患者,随访半年。比较4组患者治疗前后PEW患病率、营养不良炎症评分(malnutrition-inflammation score,MIS)及相关生化指标。结果治疗前,4组患者MIS评分(F=2.194,P=0.096)、PEW患病率(χ^(2)=0.579,P=0.901)、前白蛋白(PA)(F=0.258,P=0.855)、白蛋白(Alb)(F=0.187,P=0.905)、甲状旁腺激素(PTH)(F=0.780,P=0.509)、血红蛋白(Hb)(F=1.823,P=0.150)、β2-微球蛋白(β2-MG)(F=1.833,P=0.148)、C反应蛋白(CRP)(F=0.432,P=0.731)水平比较均无统计学差异;治疗后,D组有15例患者退出试验,C组MIS评分(C-A:t=4.391,P≤0.001;C-B:t=-2.871,P=0.006)、PTH(C-A:t=4.098,P=0.001;C-B:t=-2.551,P=0.047)、β2-MG(C-A:t=5.688,P<0.001;C-B:t=-3.207,P=0.002)、CRP(C-A:t=4.293,P<0.001;C-B:t=-2.843,P=0.006)水平低于A、B组;C组PA(C-A:t=-3.177,P=0.002;C-B:t=2.196,P=0.032)、Alb(C-A:t=-5.540,P<0.001;C-B:t=2.196,P=0.010)水平高于A、B组;C组Hb(t=-3.733,P<0.001)水平高于A组,与B组比较无统计学差异(t=1.451,P=0.152);C组PEW患病率(χ^(2)=6.465,P=0.011)低于A组,A、B 2组间PEW患病率无统计学差异(χ^(2)=2.506,P=0.113)。结论半月1次的HP治疗可以明显改善MHD患者的营养状况,降低PEW的患病率。

儿童肺炎支原体肺炎肺泡灌洗液CARDS TX、HMGB1、TLR2表达及临床意义

目的研究肺炎支原体肺炎(MPP)患儿肺泡灌洗液中社区获得性呼吸窘迫综合征毒素(CARDS TX)、高迁移率族蛋白1(HMGB1)、Toll样受体2(TLR2)表达,探讨其在MPP发病中的临床意义。方法回顾性分析55例MPP病例组及20例对照组临床资料,同时检测两组支气管肺泡灌洗液(BALF)中CARDS TX、HMGB1、TLR2、TLR4、晚期糖基化终末产物受体(RAGE)及髓样分化因子88(MyD88)表达,体外检测不同浓度CARDS TX刺激下HMGB1、TLR2的表达并进行比较。结果与对照组相比,MPP组LYM绝对值计数、PA、CD3^(+)、CD3^(+)CD4^(+)、CD3^(+)CD8^(+)、CD3-CD19^(+)、NK cell、CD19^(+)CD23^(+)明显下降,CRP、LDH、D-二聚体、IgA、IgG、IgM、CARDS TX、HMGB1、TLR2、MyD88均升高,差异有统计学意义(P<0.05或0.001)。CARDS TX刺激后HMGB1、TLR2的表达升高,且呈正相关,差异有统计学意义(P<0.001)。结论CARDS TX可能通过HMGB1-TLR2-MyD88途径参与肺炎支原体(MP)的致病。

嗜线虫致病杆菌HB310菌株杀虫蛋白的纯化及活性鉴定

嗜线虫致病杆菌XenorhabdusnematophilaHB310是从河北省土壤中筛选出的一株昆虫病原线虫体内分离纯化获得的共生菌,该菌的发酵液对多种昆虫有较高的杀虫活性。利用85%饱和度的硫酸铵盐析分别获得胞内蛋白提取物和上清液中胞外蛋白提取物,生测结果表明这两种蛋白提取物中都含有胃毒素和血腔毒素。通过制备型非变性凝胶电泳对蛋白提取物进行分离和纯化,得到了3种有杀虫活性的毒素蛋白(毒素Ⅰ、毒素Ⅱ和毒素Ⅲ),胞内的毒素蛋白与分泌到胞外上清液中的毒素蛋白是同种蛋白。毒素Ⅰ和毒素Ⅱ对棉铃虫初孵幼虫有明显的胃毒活性,但没有血腔毒性;毒素Ⅲ对大蜡螟幼虫有很强的血腔毒性,LD50为0.18μg头。SDS_PAGE图谱显示毒素Ⅰ和毒素Ⅱ是由多个多肽组成的复合蛋白,而毒素Ⅲ只分离出一条多肽。毒素Ⅱ在50℃处理10min,其杀虫活性没有显著变化;70℃处理10min对毒素Ⅲ杀虫活性没有显著影响。

转Bt基因水稻Cry1Ab杀虫蛋白在水稻土中的降解

室内研究了转 Bt基因水稻克螟稻 2号和华池B6 叶片中的Cry1Ab杀虫蛋白在稻田水稻土中的降解动态。分别将克螟稻 2号的粉碎叶片和华池B6 的整张叶片埋入 3种土壤 (即老黄筋泥田、青紫泥田和黄松田 )中 ,此后每隔 6～ 10d测定土壤或叶片中的Cry1Ab含量。克螟稻 2号粉碎叶片中的Cry1Ab均以处理后的前 36d,尤其是前 6d的降解较快 ,其中在青紫泥田中最快 ,黄松田中最慢。在处理后 6～ 36d内 ,不同土壤中的Cry1Ab残留量有显著差异 ,黄松田中的明显最高 ,老黄筋泥田中的次之 ,青紫泥田中的为最低 ;此后土壤间的残留量差异逐渐缩小 ,至处理后 78d差异已不明显。土壤淹水可显著加快华池B6 叶片中Cry1Ab的降解 ,且淹水后其降解动态在不同土壤之间十分相似 ;淹水对Cry1Ab降解的促进作用仅发生在前 12d之内 ,此后多数时间内残留量在淹水与非淹水处理间无显著差异。建立了各处理中Cry1Ab降解的动力学指数方程 ,相应地得出了其降解的半衰期。还讨论了土壤有机质和微生物等因子对土壤中Cry1Ab降解的影响。

无色杆菌毒蛋白对菜粉蝶中肠的组织病理学影响

无色杆菌毒蛋白经口进入菜粉蝶幼虫体内 6、12、18、2 4h以后 ,菜粉蝶幼虫的中肠组织逐渐遭到破坏 ,开始有些肠壁细胞伸长 ,在顶端形成囊泡 ,以后囊泡和细胞核脱落进入肠腔 .处理后期临近死亡的幼虫中肠肠壁组织被严重破坏 ,仅残留基层细胞 ,并形成一些空腔 ,从而影响昆虫的正常取食 .

Bt毒素蛋白基因的PCR鉴定及定位

利用合成的cryI、cryIII和cryV基因专一性引物 ,检测了从土壤中分离到的 56株苏云金芽孢杆菌菌株所含的晶体毒素蛋白基因。含有cryI基因的有 7株 ,含有cryIII基因的有 2株 ,同时含有cryI和cryV基因的有 2 1株。斑点杂交及Southern杂交分析表明 ,cryV基因定位于 1 50MD的大质粒上。

转Bt基因棉不同品种杀虫蛋白季节性表达及其对棉铃虫的控制作用

实验室条件下研究了不同品种转Bt基因棉花对棉铃虫抗性的差异、不同生育期棉花抗虫性的变化及Bt毒蛋白季节性表达。结果显示,不同品种转Bt基因棉在不同发育阶段,其棉叶均有阻止棉铃虫取食的作用,表现了明显的抗性,但其抗性强弱存在明显差异。DP 99B抗性表现最好,Hanza154抗性相对较差,6 d平均校正死亡率分别为89.23%和75.91%。转基因棉不同品种对棉铃虫的抗性在时间上呈现动态变化,以植株发育阶段划分,表现为蕾期>盛花期>花铃期>铃期。ELISA测定结果表明,Bt毒蛋白在转基因棉不同器官中的含量差异显著,叶片中Bt毒蛋白的表达量显著大于其它组织器官。随着棉花生长发育的推进,叶片(鲜重)中Bt毒蛋白含量则明显降低,表现为七叶期>三叶期>蕾期>铃期>花铃期。

转Bt基因作物Bt毒蛋白在土壤中的安全性研究

商业化的转Bt基因作物获准在田间大面积种植 ,使其释放的Bt毒蛋白对土壤生态系统的生态风险性问题成为人们关注的焦点 .本文综述了转Bt抗虫作物以植株残体、根系分泌物、花粉等形式释放的Bt毒蛋白通过田间耕作等方式进入土壤后的一些安全性问题 ,包括土壤活性颗粒对Bt毒蛋白的吸附作用 ,Bt毒蛋白在土壤中的杀虫活性、存留 。

转Bt基因棉Bt毒蛋白表达量的时空变化

采用抗体夹心 EL ISA技术 ,对转 Bt基因抗虫棉植株中 Bt毒蛋白含量进行了测定。结果表明 ,Bt基因在所有检测到的器官中均有表达 ,但是不同器官中的 Bt毒蛋白含量明显不同。在苗期全展功能叶中 Bt毒蛋白含量最高 ,根、茎和叶柄中 Bt毒蛋白含量较低 ;在花铃期当日开花的子房中 Bt毒蛋白含量较高 ,雌雄蕊中 Bt毒蛋白较低 ,花瓣及苞叶 Bt毒蛋白含量最低。表明 Bt基因在不同器官中的表达强度存在差异。不同生育期的功能叶中 Bt毒蛋白含量差异显著 ,Bt毒蛋白含量在苗期叶片中最高 ,蕾期次之 ,花铃期最低。随着棉花生长发育进程的推进 ,Bt基因在叶片中的表达强度逐渐减弱。Bt基因在棉花体内的表达随着器官的不同、生育时期的不同而表现出时空动态变化。这可能是人们所观察到的转 Bt基因抗虫棉对棉铃虫抗性呈时空动态变化的根本原因。