Novel Experimental Strategy for High Resolution AFM Imaging of Membrane-Associated Bacterial Toxins

Bacterial pore-forming toxins(PFTs) are essential virulence factors of many human pathogens. Knowledge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy(AFM) has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O(PFO), as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear,bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.

Generation of Reactive Oxygen Species in Different Fractions of the Coelomocytes of Holothurian Eupentacta fraudatrix in Response to the Thermostable Toxin of Yersinia pseudotuberculosis in Vitro

Pure fraction (92%-95%) of phagocytes (FP) and a mixture of amoebocytes(62%) and morula cells (38 %) FPMC of the holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirota) were obtained by using ficoll verographine step gradient. Basal production of reactive oxygen species (ROS) in FP quantified by using reduction of nitroblue tetrazolium (NBT) was more than twice that in FPMC. Thermostable toxin of Yersinia pseudotuberculosis (TST) at different concentrations ( 0.2; 0.5; 2.5 μg/ml, but not 0.1 μg/ml) stimulated NBT reduction in FPMC after 24 h incubation. In FP, TST at concentrations of 0.1 and 0.2 μg/ml inhibited and at concentrations of 0.5 and 2.5 μg/ml stimulated NBT reduction after 24 h incubation. Maximal effect was observed in FP and FPMC at TST concentrations of 0.5 and 0.2 μg/ml, respectively. Addition of catalase (0.7 μg/ml) to the cells treated with TST (2.5 μg/ml) was followed by a decrease in NBT reduction compared to that under toxin treatment alone. TST stimulated superoxide dismutase activity in concentration dependent manner (maximum at 0.5 μg/ml concentration in FP) after 24 h treatment, and this stimulation was prevented by a commercial catalase. Plant lectin concanavalin A stimulated NBT reduction more than 5 fold in FPMC compared to the control. With addition of TST, lectin stimulated ROS to lesser extent than that with lectin alone. When catalase, TST, and lectin were added into the FPMC simultaneously, ROS increase was similar to that under lectin treatment alone. On the whole, data obtained indicated that ROS generation in holothurian coelomocytes especially occurs in both stimulated and not stimulated phagocytes, and that changes in ROS production by these cells may be one of the mechanisms of antibacterial protection of holothurians.

CLINICAL APPLICATION OF BOTULINUM TOXIN TYPE B IN MOVEMENT DISORDERS AND AUTONOMIC SYMPTOMS

Objective To evaluate efficacy and safety of botulinum toxin type B (BTX-B) in treatment of movement disorders including blepharospasm, oromandibular dystonia, hemifacial spasm, tremor, tics, and hypersecretory disorders such as sia-lorrhea and hyperhidrosis. Methods A retrospective study of BTX-B injections in treatment of 58 patients with various neurological disorders was performed. The mean follow-up time was 0.9 ± 0.8 years. Results of the first and last treatment of patients with at least 3 injection sessions were compared. Results The response of 58 patients to a total of 157 BTX-B treatment sessions was analyzed. Of the 157 treatment sessions, 120 sessions (76.4%) resulted in moderate or marked improvement while 17 sessions (10.8%) had no response. The clinical benefits after BTX-B treatment lasted an average of 14 weeks. Of the 41 patients with at least 3 injection ses-sions (mean 10 ± 8.6), most patients needed increased dosage upon the last session compared to the first session. Nineteen patients (32.8%) with 27 sessions (17.2%) reported adverse effects with BTX-B treatment. Conclusions Though most patients require increased dosage to maintain effective response after repeated injections, BTX-B is an effective and safe treatment drug for a variety of movement disorders, as well as drooling and hyperhidrosis.

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.

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.

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

Role of bacteria in carcinogenesis, with special reference to carcinoma of the gallbladder

Carcinoma of the gallbladder (CaGB) is the fifth commonest gastrointestinal tract cancer and is endemic in several countries. The interplay of genetic susceptibility, infections, and life style factors has been proposed to be responsible for carcinogenesis of gallbladder. Persistence of infection leading to chronic inflammation, and production of certain toxins and metabolites with carcinogenic potentials, by certain bacteria has been speculated to be involved in the transformation of the gallbladder epithelium. Therefore, any bacteria that have evolved to acquire both of the above carcinogenic mechanisms can cause cancer. Salmonella typhi has been found to be prominently associated with CaGB. Chronic typhoid carriage (persistence) and production of mediators of chronic infl ammation and a genotoxic toxin (cytotoxic distending toxin, CdtB) are also known for this bacterium. Furthermore, the natural concentrating function of the gallbladder might amplify the carcinogenic effect of the mediators of carcinogenesis. In addition to S. typhi, certain species of Helicobacter (H. bilis and H. hepaticus) and Escherichia coli have also been implicated in carcinogenesis. As the isolation rate is verypoor with the presently available culture techniques, the existence of bacteria in a viable but non-cultivable state is quite likely; therefore, sensitive and specif ic molecular techniques might reveal the etiological role of bacterial infection in gallbladder carcinogenesis. If bacteria are found to be causing cancers, then eradication of such infections might help in reducing the incidence of some cancers.

超声血流定量参数结合BRAF对桥本甲状腺炎合并甲状腺癌的诊断价值

目的分析超声血流定量参数结合鼠类肉瘤滤过性毒菌致癌同源体B(BRAF)基因突变检测对桥本甲状腺炎(HT)合并甲状腺癌的诊断价值。方法以2020年6月至2022年6月于南京医科大学附属逸夫医院就诊的HT合并甲状腺结节患者86例为研究对象,依据病理检查结果,将合并甲状腺癌的患者为恶性组(n=26),将合并甲状腺良性结节患者为良性组(n=60)。恶性组患者中依据淋巴结转移情况分为转移组(n=10)和未转移组(n=16)。所有研究对象均接受彩色多普勒超声检查及BRAF基因突变检测,对比良、恶性组患者搏动指数(PI)、阻力指数(RI)、血流频谱收缩期峰值速度(S)、舒张末期血流速度(D)、收缩期峰值速度与舒张末期血流速度比值(S/D)、BRAF基因突变情况;对比转移组与未转移组患者彩色多普勒血流显像(CDFI)血流检出率、2~3级血流信号检出率及BRAF基因突变情况,以受试者工作特征(ROC)曲线评价上述血流参数与BRAF基因突变对HT合并甲状腺癌的诊断价值,并评估上述指标及BRAF基因突变对甲状腺癌淋巴结转移的诊断价值。结果恶性组患者的PI、RI、S、D、S/D及BRAF基因突变阳性率高于良性组(P<0.05);转移组患者的CDFI血流检出率、2~3级血流信号检出率及BRAF基因突变阳性率高于未转移组(P<0.05)。ROC曲线分析结果显示,RI、PI、S、D、S/D及BRAF基因突变联合评估HT合并甲状腺癌的曲线下面积(AUC)为0.951,敏感度为88.46%,特异度为93.33%,联合评估效能优于各指标单独评估;同时CDFI血流检出率、2~3级血流信号检出率及BRAF基因突变联合评估甲状腺癌淋巴结转移的AUC为0.938,联合评估效能优于各指标单独评估。结论超声血流定量参数结合BRAF基因突变对HT合并甲状腺癌及淋巴结转移均具有一定的评估价值,且各指标联合评估效能更佳。

基于癌毒-态靶理论消癌解毒类靶方治疗老年弥漫大B细胞淋巴瘤疗效观察

目的基于癌毒-态靶理论探讨消癌解毒类靶方和淋巴瘤中医证候量表在初诊老年弥漫大B细胞淋巴瘤免疫化疗期的临床应用价值。方法选取2022年8月—2023年8月在南京中医药大学附属医院及江苏省人民医院初诊的老年弥漫大B细胞淋巴瘤患者60例作为研究对象,采用简单随机化法将患者分为治疗组和对照组,每组30例。对照组采用R-CHOP方案化疗,治疗组采用R-CHOP方案化疗联合消癌解毒类靶方口服,2组均以21 d为1个化疗周期,治疗4个化疗周期。观察比较2组治疗前后淋巴瘤中医证候量表中单项症状评分及证候总积分、中文版欧洲癌症研究与治疗协会生活质量调查问卷(EORTC-QLQ-C30)评分及治疗4个化疗周期后的临床疗效、中医证候疗效和治疗期间不良反应发生情况。结果治疗组28例、对照组29例完成研究。治疗后,治疗组皮下肿块、神疲乏力、痛有定处、盗汗、纳差、便秘评分及证候总积分均明显低于对照组(P均<0.05);治疗后2组EORTC-QLQ-C30量表中躯体功能、角色功能、社会功能、情绪功能、认知功能评分均较治疗前明显升高(P均<0.05),且治疗组躯体功能、角色功能、情绪功能评分均明显高于对照组(P均<0.05)。治疗组客观缓解率为85.7%(24/28),对照组为72.4%(21/29),2组比较差异无统计学意义(P>0.05);治疗组中医证候总有效率明显高于对照组[92.9%(26/28)比27.6%(8/29),P<0.05]。治疗组胃肠道反应发生率明显低于对照组[10.7%(3/28)比34.5%(10/29),P<0.05]。结论消癌解毒类靶方联合免疫化疗可明显改善初诊老年弥漫大B细胞淋巴瘤能患者的中医证候,提高患者生活质量,减轻化疗胃肠道反应,基于癌毒理论的中医证候量表应用于老年弥漫大B细胞淋巴瘤中医药干预的疗效评价具有临床实际意义。

艰难梭菌毒素B2型蛋白重组表达及活性分析

目的 构建艰难梭菌高毒力株2型毒素B(TcdB2)的枯草芽胞杆菌工程株,获得高效表达的生物活性TcdB2蛋白。方法 以ST1/RT027型菌株基因组DNA为模板,通过PCR扩增获得TcdB2全长基因片段,构建到PHT01载体中,转化枯草芽胞杆菌WB800N菌株,进行原核表达获得TcdB2全长蛋白,进一步通过细胞毒性试验验证重组TcdB2的生物活性。结果 成功构建了高效表达TcdB2的枯草芽胞杆菌工程株,并获得具有细胞毒性的重组蛋白TcdB2。结论 具有生物活性的重组TcdB2蛋白,为进一步研究艰难梭菌高毒力株的致病机制和作为疫苗候选靶标等奠定了基础。

隐丹参酮调节Rac1/AKT/NF-κB信号通路对脓毒症大鼠肠损伤的影响

目的:探究隐丹参酮(CRY)调节Ras相关C3肉毒素底物1(Racl)/丝氨酸-苏氨酸蛋白酶(AKT)/核转录因子-κB(NF-κB)信号通路对脓毒症大鼠肠损伤的影响。方法:建立脓毒症大鼠模型。大鼠分为Control组、Model组、隐丹参酮高、中、低剂量组(7.0mg·kg^(-1)·d^(-1)CRY-H组、14.0mg·kg^(-1)·d^(-1)CRY-M组、28.0mg·kg^(-1)·d^(-1)CRY-L组)和隐丹参酮高剂量+NF-κB通路激活剂组(28mg·kg^(-1)·d^(-1)CRY-H+5 mg·kg^(-1)·d^(-1)PMA组),每组12只,Control组与Model组使用同等剂量生理盐水进行处理,每天1次,连续21d。用试剂盒检测脓毒症大鼠血清炎症因子、氧化应激、肠黏膜屏障指标水平;HE染色观察大鼠肠组织病理形态;免疫组化和免疫印迹法分别检测大鼠肠组织中ZO-1、occludin和通路蛋白表达。结果:与Control组比较,Model组黏膜绒毛排列紊乱,部分黏膜脱落、坏死,大量炎细胞浸润,组织病理评分较高,血清TNF-α、IL-1β、MDA、内毒素、D-乳酸水平上升,SOD水平降低,Rac1、p-AKT/AKT、p-NF-κB/NF-κB表达上调,ZO-1、occludin下调表达(P<0.05);与Model组比较,CRY-L、CRY-M、CRY-H组肠黏膜绒毛相对完整,炎细胞浸润减轻,组织病理评分降低,血清TNF-α、IL-1β、MDA、内毒素、D-乳酸水平降低,SOD水平上升,Rac1、p-AKT/AKT、p-NF-κB/NF-κB表达下调,ZO-1、occludin上调表达(P<0.05);与CRY-H组相比,CRY-H+PMA组肠黏膜细胞有较多炎细胞浸润,组织病理评分升高,血清TNF-α、IL-1β、MDA、内毒素、D-乳酸水平升高,SOD水平降低,Rac1、p-AKT/AKT、p-NF-κB/NF-κB表达上调,ZO-1、occludin下调表达(P<0.05)。结论:隐丹参酮抑制Rac1/AKT/NF-κB信号通路改善脓毒症大鼠肠损伤。

超高效液相色谱-串联质谱法测定发酵乳制品中米酵菌酸和异米酵菌酸

该研究采用液液萃取技术结合超高效液相色谱-串联质谱法对发酵乳制品中的米酵菌酸和异米酵菌酸两种细菌毒素进行监测分析,以防控细菌毒素中毒事件的发生。以发酵乳制品为基质,通过考察提取试剂、净化剂和盐析剂以及仪器参数对两种细菌毒素测定效果的影响,确定前处理条件为采用90%丙酮-1%甲酸溶液涡旋提取,高速离心浓缩,经0.22μm滤膜过滤后进行检测。在最佳条件下,米酵菌酸和异米酵菌酸在1~200 ng/mL范围内线性关系良好,相关系数(r)均大于0.999,检出限分别为0.075、0.11μg/kg,在3个加标水平下的回收率为90.8%~106%,相对标准偏差为0.80%~6.1%。该方法相较于其他方法更高效便捷,且无需前处理净化操作,展现了更佳的提取率、更高的灵敏度及更强的准确性,适用于发酵乳制品尤其是批量样品中米酵菌酸和异米酵菌酸的测定。

霍乱肠毒素B亚单位在转基因番茄果实中特异性表达及其免疫原性的研究

利用番茄果实特异性启动子-E8启动子,将霍乱肠毒素B亚单位基因(ctb)用根癌农杆菌浸润法转入番茄植株,并使其在果实中特异表达,然后对试验小鼠进行口服免疫,研究转基因番茄果实的免疫原性。结果表明,ctb基因在14株番茄植物基因组中被证实有整合,并在其中2株的番茄果实中有特异性表达,最高表达量分别为455和385 ng·g-1FW,口服免疫后的小鼠血液和肠道粘膜中均检测到抗CTB抗体。表明获得了在番茄果实中特异、高效表达霍乱肠毒素B亚单位基因(ctb)的植物口服疫苗候选植株。

霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达

目的构建霍乱弧菌肠毒素B亚单位(Cholera toxin B subunit,CTB)基因的大肠杆菌表达重组质粒,并观察其在大肠杆菌和双歧杆菌中的表达。方法从pBI121质粒PCR扩增获得CTB基因片断,克隆到大肠杆菌载体pGEX-4T-1上,构建重组质粒,然后转化大肠杆菌DH5α和双歧杆菌。转化菌经IPTG诱导,然后用SDS-PAGE和Western blot方法鉴定表达的重组蛋白。结果构建了重组质粒pGEX-4T-CTB,CTB基因片段分子量约为376bp;在大肠杆菌中表达出35kD的霍乱弧菌B亚单位融合蛋白,经SDS-PAGE分析,相对分子量与文献相符,表达的蛋白约占细菌总蛋白的10%;在双岐杆菌中也能得到正确表达,表达量较大肠杆菌低,占细菌总蛋白约5%。Western blotting结果确认了该条带为CTB基因的产物。结论构建的重组质粒pGEX-4T-CTB能够在大肠杆菌及双歧杆菌中获得表达。

霍乱肠毒素B亚单位在转基因番茄中表达的研究

将霍乱肠毒素 B亚单位 (CT-B)基因及内质网引导序列 (SEKDEL)克隆到质粒 p RTL2和 p BI1 2 1中 ,分别构建植物双元表达载体 p BI-CTB和 p BI-CTBK,CT-B基因由 Ca3 5 S启动子控制表达 .采用叶盘法经根癌农杆菌介导转化番茄 (金丰 1号 ,Jinfeng1 ) ,各表达载体得到一批转基因植株 .经 PCR和 Southern blot分析表明 CT-B基因整合到了番茄基因组中 ;ELISA和 Western blot分析表明 p BI-CTB和 p BI-CTBK的转基因植株能够有效表达 CT-B多肽 ,分别占番茄叶片可溶性蛋白的 0 .0 5 5 %和 0 .0 84% .

变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合基因转基因黄瓜植株的获得及鉴定

目的:经农杆菌介导将变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合基因导入黄瓜,获得转基因黄瓜植株,为转基因可食防龋疫苗的研究提供实验基础。方法:利用双元载体pCAMBIA2301构建变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合质粒(PAcA-ctxB)的植物表达载体p2355-PAcA-CTB;电转化法导入农杆菌EHA105;采用叶盘转化法转化黄瓜,转基因植物经过卡那霉素抗性筛选、GUS基因染色、PCR及Southern blot杂交分析检测目的基因鉴定。结果:成功得到转基因黄瓜植株,卡那霉素抗性筛选、GUS基因染色、PCR及Southern blot杂交分析检测证实目的基因PAcA-ctxB已整合至黄瓜基因组中。结论:获得了含有变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合基因的转基因黄瓜植株,为转基因可食防龋疫苗的进一步研究提供了实验基础。

ctB和ure I双基因融合表达及其免疫特性分析

目的构建霍乱毒素B亚单位(CtB)和幽门螺杆菌尿素膜通道蛋白(UreI)融合的原核表达质粒pET32a(+)ctB/ureI,并初步研究融合蛋白CtB/UreI的表达特性和免疫特性。方法PCR从pUC18ctB中克隆ctB基因,定向在pET32a(+)/ureI的ureI基因5′端插入ctB基因,构建ctB和ureI双基因原核表达质粒pET32a(+)ctB/ureI,转该质粒于E.coliBL-21(DE3),经酶切和序列分析鉴定工程菌。IPTG诱导表达,HP-His亲和层析纯化,SDS-PAGE和Gel-ProAnalizer4分析,重组蛋白免疫BALB/c小鼠。用Westernblot和ELISA分析重组蛋白的免疫特性。结果工程菌含完整的ctB和ureI基因,与相对应基因的序列同源性分别为100%。在22℃,1mmol/LIPTG诱导4h后,重组蛋白的表达占菌体总蛋白12%,亲和层析纯化后蛋白纯度为94.3%。Westernblot表明重组蛋白分别能与相应的抗体反应,该蛋白免疫小鼠后能产生相应的IgG抗体。结论成功构建了能表达CtB/UreI蛋白的大肠杆菌表达菌株。对融合蛋白表达和纯化后,初步证明了该重组蛋白有CtB和UreI的双特异反应原性和免疫原性,为研究新型幽门螺杆菌疫苗奠定了坚实的基础。

基于火毒病机研究急性脑梗死重症大鼠皮层的核转录因子κB、c-fos蛋白表达及中药干预效应

目的基于“火毒”病机观察急性脑梗死重症大鼠皮层的核转录因子KB（NF-KB）、c-fns蛋白表达及中药干预效应。方法以Longa的方法略作改良制作鼠左侧大脑中动脉阻塞再灌注模型，选择Bederson评分〉2分的重症急性脑梗死大鼠，对照单纯扶正方药及单纯解毒方药，观察扶正解毒方药对急性脑梗死重症大鼠的宏观表征、NF-KB、c-fos蛋白表达的影响。结果（1）假手术组脑组织细胞核明显呈淡淡的浅棕黄色，模型各组细胞核着色呈现明显的棕黄色，以缺血24小时-48小时为主要变化，给予扶正药、解毒药和扶正解毒药后，神经核着色较浅，其中以扶正解毒药改善效果较为明显，各药物组着色仍比假手术组明显。脑组织细胞浆呈现相同趋势；（2）缺血各模型组大鼠，缺血侧c-fos和NF-KB蛋白表达明显高于假手术组（P〈0．01），扶正药和解毒药治疗组c-fos和NF-KB在缺血24小时-48小时时蛋白表达明显低于模型组（P〈0．05～0．01），扶正解毒药治疗组在缺血各时间点c-fbs和NF-KB的蛋白表达均明显降低（P〈0．01）。结论重症急性脑梗死大鼠的NF-KB、c-fos蛋白明显升高；扶正解毒中药治疗重症脑梗死大鼠可能与调节NF-KB、c-fos蛋白表达水平有关。

大肠杆菌志贺毒素stxB融合基因的构建及其高效表达

利用 PCR方法 ,从肠出血性大肠杆菌 O15 7∶ H7的基因组 DNA中 ,分别扩增出 Stx1B和 Stx2 B亚基的结构基因片段 ,然后再通过 PCR将这 2个片段连接起来 ,并定向克隆到表达质粒 p ET2 8a的 Nco 和 Eco R 位点之间 ,构建了重组表达质粒 p MB1。将重组质粒转化到宿主菌 BL2 1(DE3)中 ,对重组菌株 BL2 1(p MB1)用 IPTG进行诱导表达 ,并对最佳诱导时间进行了探索。 SDS- PAGE电泳检测结果表明 ,重组菌株表达出了 16 30 0的目的蛋白 Stx2 B- L-Stx1B;经薄层扫描分析表明 ,表达量约占菌体总蛋白的 6 8.4 % ,且目的蛋白为包涵体表达 ,表达量约占细菌沉淀的84 .6 %。研究还表明 ,未用 IPTG诱导的 BL2 1(p MB1)菌株也可有少量目的蛋白的基础表达。不同诱导时间的结果表明 ,在 3～ 7h的诱导时间内 。